B-lymphocyte activation with an extract of Nocardia brasiliensis.

An extract from the pathogenic actinomycete Nocardia brasiliensis was mitogenic for murine lymphocytes. This deoxyribonucleic acid-synthetic response of whole spleen cells peaked after 48 h in culture at concentrations of Nocardia extract ranging from 10 to 200 micrograms/ml. The extract appeared to be a mitogen for B lymphocytes since cultures of spleen cells from congenitally athymic nude (nu/nu) mice and of antithymocyte serum plus complement-treated spleen cells from conventional (+/+) mice responded as well as untreated spleen cells from normal +/+ mice. Furthermore, thymocytes did not respond mitogenically to the extract. Mitogenic responses were stimulated in spleen cells from H-2(a), H-2(b), H-2(d), and H-2(k) mice, including lipopolysaccharide-nonresponder C3H/HeJ mice. This Nocardia extract also stimulated polyclonal B-cell activation to the hapten trinitrophenyl, serum protein human gamma globulin, and several mammalian erythrocytes in cultures of cells from both euthymic and nude mice. Additionally, the requirement for helper T cells in the primary in vitro immune response to sheep erythrocytes could be circumvented by the addition of this Nocardia extract. These results indicate that an extract from the pathogen N. brasiliensis can nonspecifically activate murine B lymphocytes and raise the possibility that polyclonal activation of B lymphocytes may contribute to the pathogenesis of nocardiosis.     

Suppression of antibody response by group A streptococcal pyrogenic exotoxin and characterization of the cells involved.

The effect of purified streptococcal pyrogenic exotoxins (SPE) on the antibody response to sheep erythrocytes was studied in cultures of mouse spleen cells. Purified SPE types A, B, and C shared the ability to suppress the day 4 direct plaque-forming cell response when added to cultures. SPE A and C were most suppressive at concentrations of 0.1 to 1 ng per culture, while SPE B was active at 1 microgram per culture. Pretreatment of mice with SPE A, 3 h before removal of their spleens for culture, also produced suppression. Cell populations were separated from spleens of normal and toxin-treated mice and recombined in culture to test the cellular site of action of SPE immunosuppression. When nonadherent cells (lymphocytes) and adherent cells (macrophages) from control and SPE-treated mice were separated and recombined, the plaque-forming cell response depended on the source of lymphocytes. Macrophages from toxin-treated mice functioned normally in the presence of control lymphocytes. In a further experiment, toxin pretreatment failed to suppress the plaque-forming cell response of spleen cells that were T-cell depleted and reconstituted with control thymocytes. When the T lymphocytes were removed from toxin-treated spleen cell suspensions, the remaining cells were able to respond normally to antigen if normal helper T cells were provided. The results suggest that the suppressive activity of SPE on antibody production is mediated by altered activity of T lymphocytes.     

In Vitro and in Vivo Stimulation of Murine Lymphocytes by Human Respiratory Syncytial Virus Strains

Respiratory syncytial virus (RSV) strains of subtype A (A2, WV9894, and WV12138) and of subtype B (WV1293, WV4843, and WV6873) are mitogenic in vitro for unprimed BALB/c spleen cells. The virus also triggered splenocytes in vitro to secrete immunoglobulins. Plaque-purified and UV-irradiated materials of both RSV subtypes produced comparable levels of DNA synthesis. Infectious materials of both subtypes also induced pronounced responses. Lymphocyte activation with UV-inactivated RSV strain A2 was dose-dependent and maximal responses occurred after 4-5 days of incubation. The virus preparations were mitogenic for spleen cells depleted of T lymphocytes by treatment with anti-Thy 1.2 and complement and for lymphocytes of congenitally athymic mice (nu-nu). They were also mitogenic for highly purified T lymphocytes separated by panning of spleen cells on anti-mouse Ig-coated Petri dishes, suggesting that both B and T lymphocytes respond to the mitogenic activity of RSV. Moreover, mice infected intranasally with RSV strain A2 generated local as well as peripheral cellular and humoral responses.     

Altered neutrophils in mice immune to experimental Naegleria amoebic meningoencephalitis

Naegleria fowleri is the cause of primary amoebic meningoencephalitis in man. The mouse is considered to be a suitable experimental model for this disease. The data presented shows that blood neutrophils from N. fowleri immune mice (immunised) that had received a 'recall' amoeba antigen had altered responses compared with those from similarly treated normal mice. The neutrophils from immune animals showed increased basal levels of oxygen-dependent respiratory activity, measured by the chemiluminescence response. These neutrophils also showed increased responses to formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), phorbol myristate acetate (PMA), and opsonized N. fowleri. The results are consistent with previous data suggesting that the neutrophil (and its activation) is important in defence against N. fowleri.     

Induction of suppressor cells in Japanese encephalitis virus infected mice

Adoptive transfer of spleen cells obtained from mice primed with Japanese encephalitis virus (JEV) suppressed IgM antibody plaque forming cells (PFC) against JEV in the spleen. Similar suppression of PFC was also shown in vitro by adding primed spleen cells to JEV-stimulated spleen cell cultures. The suppressor activity appeared sharply in the third week after priming and persisted up to 6 weeks. By using various cell separation procedures it was found that the suppressor activity resided in the T cell enriched fraction and not in B cells or macrophages. Sensitivity of the cells to treatment with anti-Thy 1.2 antiserum and complement confirmed that suppressor cells were T lymphocytes. It was noted that the suppression was effective against dengue virus antigen also. Our findings thus show generation of suppressor T lymphocytes in JEV-infected mice.     

On evasion of Trypanosoma cruzi from the host immune response. Lymphoproliferative responses to trypanosomal antigens during acute and chronic experimental Chagas' disease.

The ability of spleen cells taken from mice infected with Trypanosoma cruzi to proliferate after stimulation with specific trypanosomal antigens was investigated during the acute and chronic phases of the disease. Lymphoproliferation was minimal or undetectable during the acute period whereas the chronic phase was characterized by significant responses over a wide range of antigen concentration. Transfer of infected mouse spleen cells to cultures of splenocytes from chronically infected animals failed to modify the response of the latter to antigenic stimulation as measured by DNA synthesis. Furthermore, the responses of infected mouse spleen cells collected during the acute period and freed of Lyt 2.1-bearing lymphocytes, a subclass known to contain the suppressor T cells, did not differ significantly from those of untreated aliquots of the same cell suspensions. These results, together with the fact that the T-cell compartment of the spleen was severely depleted during the acute but not the chronic stage of the infection, suggest that the impaired immunological responsiveness of acutely infected mice may be due in part to the absence or marked reduction of responder and/or accessory T lymphocytes. An active role for suppressor T cells in the reduced response to trypanosomal antigens by lymphocytes from mice in the early, acute phase of T. cruzi infection is not supported by the present observations.     

Induction of suppressor cells in Japanese encephalitis virus infected mice.

Adoptive transfer of spleen cells obtained from mice primed with Japanese encephalitis virus (JEV) suppressed IgM antibody plaque forming cells (PFC) against JEV in the spleen. Similar suppression of PFC was also shown in vitro by adding primed spleen cells to JEV-stimulated spleen cell cultures. The suppressor activity appeared sharply in the third week after priming and persisted up to 6 weeks. By using various cell separation procedures it was found that the suppressor activity resided in the T cell enriched fraction and not in B cells or macrophages. Sensitivity of the cells to treatment with anti-Thy 1.2 antiserum and complement confirmed that suppressor cells were T lymphocytes. It was noted that the suppression was effective against dengue virus antigen also. Our findings thus show generation of suppressor T lymphocytes in JEV-infected mice.     

Dengue virus-induced thymus-derived suppressor cells in the spleen of mice.

Adoptive transfer of spleen cells obtained from mice given three weekly i.p. doses of dengue type 2 virus (DV) suppressed DV antigen-specific antibody secretion as detected by the Jerne plaque technique. This suppression was produced by non-glass-adherent cells but not by glass-adherent cells. Immune spleen cells depleted of macrophages by carbonyl iron treatment had higher suppressor activity. Immune spleen cell homogenate could transfer the activity equally well. The immune spleen cells were separated into T and B lymphocytes by a nylon wool column. B lymphocytes had no suppressor activity; almost all the suppressor activity was present in T lymphocytes. Thus, macrophages and B lymphocytes had no suppressor activity; it was mediated by T lymphocytes through soluble factors.     

Sendai virus glycoproteins are T cell-dependent B cell mitogens

UV-inactivated Sendai virus is mitogenic for murine splenocytes, whereas infectious Sendai virus kills spleen cells in vitro. The isolated hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of Sendai virus are also mitogenic for cultured mouse spleen cells. A mixture of these glycoproteins (1 microgram/well) gives maximum stimulation 96 h after culture initiation. Viral proteins remaining insoluble after Triton X-100 extraction are also mitogenic for mouse spleen cells, with maximum stimulation occurring at 72 h after culture initiation with 1 to 5 microgram/well. On the basis of protein concentration, the HN and F glycoproteins are approximately three times more mitogenic than the Triton X-100-insoluble material. The mitogenic response of the HN and F glycoproteins has two components, a T cell-independent B cell proliferation, which is less than one-half of the total stimulation observed, and a T cell-dependent B cell proliferation. In contrast, the Triton X-100-insoluble material is a T cell-dependent B cell mitogen. Purified T lymphocytes do not respond to the mitogenic signal of either HN-F or Triton X-100-insoluble material.     

Induction of suppressor macrophages in mice by Fusarenon-X

Intraperitoneal injection of Fusarenon-X into BALB/c mice, a mycotoxin produced by Fusarium nivale Fn 2B, depressed polyclonal antibody formation of splenic lymphocytes in response to pokeweed mitogen (PWM). This inhibitory activity was found to reside in the surface immunoglobulin-negative spleen cell fraction of Fusarenon-X-treated mice, sIg-(FX), which comprised mainly T lymphocytes and smaller number of non-lymphocytic cellular elements. However, reconstitution experiments for in vitro antibody formation provided evidence that T lymphocytes from Fusarenon-X-treated mice, T(FX), which were separated from non-lymphocytic cells by use of carbonyl iron/magnet, were as effective as T lymphocytes from normal mice, T(N), in supporting antibody formation. Furthermore, addition of non-lymphocytic cells, NL(FX), or adherent cells, AD(FX), prepared from spleen cells of Fusarenon-X-treated mice to normal spleen cells strongly inhibited in vitro antibody formation against PWM or a bacterial lipopolysaccharide (LPS). These results strongly indicated that Fusarenon-X induced non-lymphocytic suppressor cells in the spleen of the treated mice which had features in common with activated macrophages.     

Effect of infectious bursal disease on natural killer cell activity and mitogenic response of chicken lymphoid cells: role of adherent cells in cellular immune suppression.

A pathogenic isolate of infectious bursal disease virus (IBDV) caused persistent and extensive lesions in the bursa but mild and transient lesions in the thymuses of chickens of lines 63 and P. The effect of IBDV on two cellular immune functions, namely, natural killer cell cytotoxicity and mitogenic response, was studied. The natural killer cell activity was not consistently influenced, but the virus, during the first 2 weeks of infection, caused transient depression of the blastogenic response of spleen cells to phytohemagglutinin. Studies on mitogenic hyporesponsiveness revealed that the functional impairment was mediated by a suppressor cell that shared several characteristics with macrophages; i.e., the suppressor cell was adherent to plastic, was phagocytic, and resisted treatment with antithymocyte and antibursa cell sera. Removal of suppressor cells from the spleens of virus-infected chickens resulted in restoration of the mitogenic response of cells. Further, in mixing experiments, the suppressor cell isolated from the spleens of virus-infected chickens also inhibited the mitogenic response of normal spleen cells. We concluded that reduced mitogenic response of lymphocytes in IBDV-infected chickens was not due to a lack of functional T-cells, as suggested previously by others, but was due to macrophage-like suppressor cells. The suppressor cells, although present in certain normal chickens, became activated during early stages of IBDV infection.     

紫色杆菌LPS对小鼠脾细胞免疫活性的抑制作用

细菌内毒素或脂多糖(LPS)是机体内强烈的免疫调节剂。天然低毒性紫色杆菌LPS 体内处理小鼠,能促进脾细胞的分化、增殖,但降低脾细胞对 Con A 和同种细菌 LPS 的反应性,抑制混合淋巴细胞反应(MLR)和自然杀伤(NK)细胞的活性。LPS 处理供体小鼠还抑制其脾细胞在 F_1 小鼠内诱导的移植物抗宿主反应。应用混合培养方法,在 Con A和 LPS 诱导的淋巴细胞转化反应中分别检测到非特异性抑制细胞活性,但在MLR和NK 活性测定中未发现抑制细胞的存在。上述结果说明 LPS体内抑制T、B 淋巴细胞功能和 NK细胞活性,而这种抑制作用除由抑制细胞介导外,还存在其它尚未明瞭的机理。     

Mitogenic activity of staphylococcal peptidoglycan.

Staphylococcus aureus peptidoglycan displayed a marked dose-dependent mitogenic activity for mouse splenocytes and human peripheral blood lymphocytes in vitro, as measured by increased [3H]thymidine incorporation. Similarly it was mitogenic for athymic nude mouse spleen cells, whereas no blastogenic effect was observed in T cell-enriched and B cell-depleted mouse lymphocyte cultures. These data demonstrate that peptidoglycan-responding cells in mouse spleen cell cultures are B lymphocytes.     

Mice naturally tolerant to C5 have T cells that suppress the response to this antigen

We examined whether C5-sufficient mice which are naturally tolerant to this antigen have suppressor T cells to C5 humoral immune response. Two congenic strains of mice B10.D2 (NSN) and B10.D2 (OSN) differing only in the presence or absence of C5 were used. Irradiated (760 rds) sufficient hosts were reconstituted with a nonadherent spleen cell suspension from either sufficient or deficient mice or a mixture of both. Hemolytic C5 levels were assayed. Sufficient spleen cells appeared to prevent the drop of C5 level caused by anti-C5 antibody made by deficient spleen cells. Spleen cell suspensions from sufficient mice primed with deficient spleen cells exhibited better anti-C5 activity than normal sufficient spleen cell suspensions. This anti-C5 activity is abrogated by treatment of the NSN spleen cell suspensions obtained from NSN primed with OSN spleen cells with anti-Thy-1.2 antiserum and complement. Suppression of the humoral response to C5 failed to affect the anti-sheep red blood cell immune response. Suppressor T cells are resistant to low-dose irradiation, cortisone treatment and adult thymectomy. In contrast, they are sensitive to high doses of irradiation and both high and low doses of cyclophosphamide treatment. Thus, C5-sufficient mice, in contrast to C5-deficient mice, appear to have antigen-specific suppressor T cells which downregulate the humoral immune response to C5. In addition, we examined the relationship of these suppressor T cells to the state of tolerance in helper T cells of C5-sufficient mice. This was done in irradiated deficient mice which were repopulated with spleen cell suspensions selectively depleted of either Lyt-1+ or Lyt-2+ T cell subsets. These chimeras were challenged with murine C5 and both the primary and secondary immune response was measured by inhibition of the C5 hemolytic activity. It was found that only spleen cell suspensions of the deficient mice selectively depleted from the Lyt-2+ subset of T cells responded to the antigen both in the primary and secondary response. In contrast, either subset of T cells from the sufficient mice failed to respond. Thus, it appears that in sufficient mice helper T cells to C5 are intrinsically tolerant or physically and/or functionally deleted. In conclusion, the data suggest that both T cell compartments are unresponsive and play a role in the mechanism of tolerance to a physiologic antigen.     

Lymphocyte function in experimental african trypanosomiasis: mitogenic effects of trypanosome extracts in vitro.

Extracts of Trypanosoma brucei and Trypanosoma congolense were incubated in vitro with nonimmune lymphocytes of mice, rats, guinea pigs, and rabbits in order to test for mitogenic effects or for other characteristics of polyclonal B lymphocyte activators. Trypanosome extracts (TE) were not mitogenic for spleen cells of mice, rats, and guinea pigs in vitro, nor did the parasite extracts alter the mitogenic responses of lymphocytes from these animals to known B- and T-cell mitogens. TE also failed to induce polyclonal antibody synthesis in mouse spleen cell cultures in an in vitro antibody response system, in contrast to the effects of bacterial lipopolysaccharide, a known polyclonal B cell activator. Rabbit spleen cell and peripheral blood lymphocyte cultures, however, were stimulated by TE to undergo blastogenesis in vitro. Incubation of rabbit lymphocytes with phytohemagglutinin (PHA) and TE or anti-rabbit immunoglobulin serum and TE revealed an additive effect only in terms of the TE-plus-PHA culture responses; these findings suggest that a non-PHA responsive lymphocyte population, possibly B lymphocytes, is stimulated by TE in rabbits. The relationship of trypanosome-induced lymphocyte mitogenic stimulation to other immunological dysfunctions occurring in chronic African trypanosomiasis is discussed.     

The Induction of Suppressor T Cells by Concanavalin A is Independent of Cellular Proliferation and Protein Synthesis

Factors that govern the induction of suppressor T cells after stimulation with concanavalin A (Con A) were investigated in a two-stage culture system. Normal mouse spleen cells were incubated with Con A in the presence of a variety of drugs and then assayed for suppressive activity by means of a secondary anti-sheep erythrocyte response in vitro. The inclusion of inhibitors of mitosis (vinblastine sulphate or mytomycin C) or protein synthesis (cycloheximide or pactamycin) into normal spleen cell cultures containing Con A failed to inhibit the subsequent development of suppressor cells. Furthermore, spleen cells from mice previously irradiated with 900 rad or injected with cyclophosphamide expressed a level of suppressor activity after Con A stimulation which was equivalent to that of normal spleen cells. However, the inclusion of drugs that inhibit microtubule or microfilament function (colchicine or cytocholasin B) did prevent suppressor cell induction. Kinetic studies also revealed that significant suppressor activity was detectable in normal spleen cells after only 3 h exposure to Con A. These results indicate that the induction of suppressor T cells in this system is a maturation event involving changes in the cell membrane and is entirely independent of protein synthesis and cellular proliferation.     

Activation of murine B lymphocytes by Neisseria meningitidis and isolated meningococcal surface antigens.

Heat-killed Neisseria meningitidis was found to be a potent mitogen for mouse splenic lymphocytes. Results obtained with different cell separation techniques indicated that the bacteria acted to selectively induce proliferation of B lymphocytes. First, partial or total depletion of T lymphocytes by treatment with various anti-T-cell antisera plus complement did not affect the ability of the remaining spleen cells to proliferate in response to N. meningitidis. Second, T lymphocytes purified by affinity chromatography through an immunoglobulin-antiimmunoglobulin-coated glass bead column were unresponsive to meningococcal stimulation, even when provided with a source of macrophages (irradiated or mitomycin C-treated spleen cells). Finally, treatment of spleen cells with soy bean agglutinin showed that, whereas the soy bean agglutinin-positive population (B-enriched lymphocytes) was highly responsive to stimulation by N. meningitidis, the soy bean agglutinin-negative population (T-enriched lymphocytes) displayed only a background level of proliferation when exposed to the bacteria. Isolated meningococcal surface antigens such as lipopolysaccharide (LPS) and outer membranes also possessed mitogenic activity and induced proliferation of B lymphocytes in a dose-dependent manner. Both LPS and non-LPS components contributed to the mitogenicity of outer membranes since the addition of outer membrane preparations to spleen cells from the low LPS responder C3H/HeJ mouse strain gave rise to a high level of proliferative activity.     

Specific and Non-specific Activation of Human and Mouse Lymphocytes in Vitro by Pathogenic and Apathogenic Neisseria Strains

In vitro lymphocyte responses to gonococcal antigen (T2), apathogenic Neisseria pharyngis (APN), PPD and PHA were studied in patients with uroarthritis and in healthy controls. Only the T2 antigen induced a significantly higher DNA synthetic response in cells from patients than in controls, whereas no difference was observed with the other substances. Peak responses occurred on day 6 of culture with a concentration of 4 × 10⁷ bacteria/ml. In order to test whether lymphocyte responses were the result of specific sensitization, experiments were carried out with cord blood cells. These cells reponded to both T2 and APN with maximum activity on day 6 of culture. Since specific sensitization was ruled out in these experiments, we concluded that T2 and APN were capable of inducing a non-specific mitogenic response in cord blood cells. The mitogenic activity of T2 and APN was further elucidated in experiments with mouse lymphocytes. Both substances induced increased DNA synthesis in mouse spleen cells, with peak activity on days 2–3 of culture. Stimulation occurred in spleen cells depleted of macrophages, in anti-Thy 1.2 treated spleen cells and in Nude spleen cells, but not in thymus cell cultures. It was concluded that T2 and APN contained polyclonal B cell activating (PBA) substances, which was subsequently proved by their ability to induce a polyclonal antibody response in cell culture. The B cell subpopulation that responds to the mitogenic properties of these bacteria is normally absent from blood, present as a minor proportion of cord blood lymphocytes and present as a major cell population in spleen. Therefore, in spite of the mitogenic properties of these bacterial strains, they are useful tools for the elucidation of a specific reactivity in patients with uroarthritis.     

犬蛔虫（Toxocaracanis）对感染鼠的细胞免疫和IL－1、IL－2免疫调节作用的影响

应用犬蛔虫（Ｔ．ｃａｒｎｉｓ）的感染性虫卵口饲感染Ｃ３Ｈ／ＨｅＮ鼠，体外培养后观察脾细胞的免疫学变化。感染第１－４周的脾细胞用ＣｏｎＡ刺激，Ｔ细胞的增殖反应，ＩＬ－２的诱生均明显地受到抑制，而且感染鼠脾细胞抑制了正常鼠脾细胞对ＣｏｎＡ的反应。感染鼠的脾细胞经ＳｅｐｈａｄｅｘＧ－１０过柱后，Ｔ细胞则不显示被抑制作用。表明影响Ｔ细胞被抑制作用的是感染鼠的巨噬细胞。相反，感染第１－４周的鼠所产生的ＩｇＧ和ＩｇＭ等抗体的Ｂ细胞活性增强；用ＬＰＳ刺激巨噬细胞诱生的白介素（ＩＬ）ＩＬ－１也增多。     

Mechanisms of action of Mycobacterium bovis BCG-induced suppressor cells in mitogen-induced blastogenesis.

Spleen cells from Mycobacterium bovis BCG-infected C57B1/6 mice when cultured in vitro for 72 h elicited soluble suppressor mediators capable of nonspecifically suppressing the mitogen-induced blastogenesis of normal splenocytes. Maximal production of suppressor mediators occurred during the first 24 h in culture, and their production ceased after 72 h. Attempts to isolate the mediators from fresh nonincubated splenocytes failed. After incubation, a strong residual suppressive activity was constantly detected in cell preparations used for production of suppressor factors. Supernatants prepared from cultures of spleen cells of mice infected 14 days earlier possessed higher suppressive activity than did those obtained 28 days after infection. In contrast, the residual cellular suppressive activity increased during the course of the infection. Although the activity of soluble factors was not inhibited, the residual activity of incubated cells was highly depressed by the presence of mouse erythrocytes in the cultures. Thus, the incubated cells appear to act through a direct cell-to-cell contact with the mitogen-responding cells. Finally, the results of depletion experiments suggest that the two populations of BCG-induced suppressor cells, namely, T lymphocytes and macrophage-like cells, are able to elicit suppressor mediators and to retain thereafter suppressive activity.