Interdependence of in vitro responsiveness of cord and maternal blood lymphocytes to antigens from oral bacteria.

A group of thirty-five mothers and their babies at parturition were examined by the in vitro lymphocyte transformation test to determine sensitization by oral bacterial antigens, B-cell mitogens and dental plaque. Lymphocytes from babies of sensitized mothers with gingival or periodontal disease gave the highest frequency (70 and 63%) and magnitude (mean stimulation index of 3.4 and 3.3) of response in cultures stimulated by Actinomyces viscosus and Veillonella alcalescens. However, IgM antibodies to V. alcalescens antigen were absent from cord sera. With one exception, stimulation of lymphocytes from babies of unsensitized mothers with clinically healthy gingiva was not found with these antigens. The response of cord lymphocytes from mothers with gingival or periodontal disease to antigens from oral bacteria, as compared with the response of cord lymphocytes from mothers with clinically healthy gingiva, seemed specific, since a corresponding difference in response to unrelated antigen PPD was not found. The response of cord and maternal lymphocytes to B-cell mitogens was also determined. Maternal lymphocytes responded in the following decreasing order of effectiveness: dextran sulphate, levan, lipopolysaccharide and dextran B1355; whereas cord lymphocytes were stimulated in the reverse order of effectiveness.     

IgE synthesis in vitro during infection of mice with the nematodeNippostrongylus brasiliensis: Effects of mitogens and antigens

In vitro IgE synthesis by lymphoid cells was studied during the course of infection of mice withNippostrongylus brasiliensis. The studies involved inbred strains of mice which had been shown to be high IgE responders (A.CA, B10.M), or non-responders (Balb/c, B10.D2) to parasite antigen. In addition, F₁ hybrids of low and high responders and irradiated non-responders were studied. Infection withN. brasiliensis led to an increase in IgE synthesis in vitro which was most pronounced during reinfection of mice. Addition of mitogens e.g. pokeweed mitogen (PWM), lipopolysaccharide (LPS), concanavalin A (ConA) to the cultures induced enhancement, suppression or had no effect on IgE synthesis. Addition ofN. brasiliensis homogenate or worm culture supernatant led to a fluctuating pattern of IgE synthesis. No correlation was found between lymphocyte proliferative response to mitogen and worm antigens and IgE synthesis. Our data suggest, that PWM is more likely to enhance IgE synthesis in vitro than LPS or ConA. An enhancement is more easily observed with the cells of non-infected animals or during the early phase of infection or reinfection. The mitogen-induced increase in IgE synthesis did not exceed the values obtained during infection or reinfection.     

Cellular Immunity in Human Milk

ABSTRACT: The responses of human milk lymphocytes (MIL) to a variety of immunogenic stimuli were studied and compared to those of peripheral blood lymphocytes (PBL) from the milk donors. MIL showed a decreased proliferative response to mitogens and allogeneic leukocytes in vitro but displayed the ability to stimulate alloreactivity equivalent to PBL. Neither pretreatment with cell-free autologous milk nor co-cultured MIL were capable of suppressing the proliferative responses of PBL. Moreover, macrophages isolated from milk and pulsed with soluble antigen or allogeneic cells effectively induced proliferation by peripheral blood T cells whereas the response of milk nonadherent cells to antigen presented by peripheral macrophages was very low. MIL respond better to pathogenic enteric E. coli than PBL but not as well as PBL to Yersinia enterocolitica. Treatment of MIL with monoclonal antibodies cytotoxic for T cells abolished their response to bacterial antigens. Application of an anti HLA class II antigen monoclonal antibody to mixed lymphocyte or lymphocyte-bacteria cultures resulted in substantial inhibition of the MIL response similarly to that of PBL. The relevance of these data to the immunological needs of the neonate are discussed.     

Induction of DNA synthesis in human lymphocytes: Interaction between non-specific mitogens and antigens

Human blood lymphocytes stimulated in vitro with non-specific mitogens, such as PHA, ALS and streptolysin, responded with DNA synthesis only to certain optimal doses of the mitogens, higher and lower doses failing to stimulate the cells. With ALS only a 100-fold difference in concentration covered the entire dose range. Specific antigens on the other hand were stimulating over a 10,000-fold difference in concentration. The simultaneous stimulation of lymphocytes with two non-specific mitogens resulted in antagonistic or synergistic effects. Antagonism was found when two mitogens were added in optimal concentrations, whereas synergism occurred when both were present in suboptimal concentrations. Non-specific mitogens in high concentrations abolished induction of DNA synthesis by specific antigens, whereas they increased the response to antigen when they were added in low, by themselves non-stimulating, concentrations. The mixed lymphocyte culture reaction was found to abolish stimulation caused by non-specific mitogens added 3 days later.

The findings suggest that individual lymphocytes respond with DNA synthesis after contact with a stimulus only when a threshold number of sites on the surface has been triggered. When the number of triggered sites is increased above a certain higher threshold, the lymphocytes fail to respond. Furthermore, the lymphocytes do not discriminate between the quality of the sites triggered, but only between the number of triggered sites, disregarding the nature of the stimulus. Finally, sensitized and presumably thymus-processed lymphocytes, confronted with antigen appear to influence other non-antigen recognizing cells, presumably by releasing some factors. These factors are capable of stimulating non-antigen-recognizing cells or to make them refractory to stimulation by subsequently added mitogens.

     

Study of the NKT cell subsets with superantigen SEB-activated tolerance

AIM: To study the NKT cell subsets and their differentiation. METHODS: Splenic lymphocytes from C57BL/J mice that had received SEB treatment were collected as effector cells on the 10(th) day. The cells were cultured in medium containing ConA, LPS and IL-2 for 3 days and measured their response to mitogens and cytokine. The inhibitory action of the effector cells was examined. The effector cells were cultured with normal lymphocytes and above mitogens or cytokine for 3 day. The cells proliferation was assessed with MTT method.The NKT cell subsets among these effector cells with the tolerance function were analyzed and their differentiation sources and correlation of functions were detected by flow cytometry. RESULTS: The response of SEB-activated effector cells to ConA, LPS and IL-2 was significantly decreased compared with that of normal lymphocytes. The A values of cell proliferation were decreased from 0.80+/-0.04, 0.60+/-0.03 and 0.55+/-0.07 in control groups to 0.60+/-0.05, 0.30+/-0.05 and 0.27+/-0.04 in effector groups, respectively (P<0.01, n=3).The inhibitory ability of effectors cells against the response of normal lymphocytes to ConA, LPS and IL-2 were clearly observed. They inhibited the response of normal lymphocytes to several mitogens and cytokine. And the A values of cell proliferation were decreased to 0.26+/-0.02, 0.48+/-0.04 and 0.34+/-0.02, respectively (P<0.01, n=3). The CD4(+)NK1.1(+), CD8(+)NK1.1(+), TcRV8(+)NK1.1(+) NKT cell subsets among SEB-activated effector cells with tolerance function were significantly increased and shown that they come from T cell population. And the CD4(-)CD8(-)/NK1.1(+)CD3(+)NKT cells by ConA or SEB-activated were shown coming from NK cell population. CONCLUSION: The effector cells with tolerance function activated by superantigen SEB relate to CD4(+)NK1.1(+), CD8(+)NK1.1(+), TcRVbeta8(+)NK1.1(+) NKT cell subsets. The NKT cell subsets come from T cells. The CD4(-)CD8(-)/NK1.1(+)CD3(+)NKT cells differentiating from NK cells are not involved in the regulation of tolerance.     

Cooperative effects in mitogen- and antigen-induced responses of human peripheral blood lymphocyte subpopulations.

Human peripheral blood lymphocytes were separated into T cell-enriched and T cell-depleted fractions by E rosette sedimentation. These two fractions, as well as the unseparated lymphocyte suspension, were tested for their responsiveness to the mitogens phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) and to the antigens PPD (purified protein derivative of tuberculin) and tetanus toxoid. The response to PHA, ConA and the antigens was found to be confined to the purified T cell fraction; PWM could stimulate both purified T and non-T cells. However, the T cell response to ConA, PPD and tetanus toxoid was always decreased by 50-70%, when compared to the unseparated lymphocytes. Addition of monocytes could restore the T cell response. In the response to PHA and tetanus toxoid, the (primarily unresponsive) non-T cell fraction could be recruited into proliferation by gamma-irradiated T cells. Moreover, in the response to tetanus toxoid, lymphocytes (T as well as non-T) from a nonimmune individual could be recruited into proliferation by gamma-irradiated immune T cells.     

连翘提取物对小鼠T淋巴细胞体外活化与增殖的影响

目的:分析连翘(FS)提取物对小鼠淋巴结T细胞的体外活化与增殖的影响,初步探讨其免疫抑制作用机制.方法:无菌分离小鼠淋巴结细胞,加入多克隆刺激剂刀豆蛋白A(ConA)进行刺激,利用荧光标记的单克隆抗体(mAb)染色结合流式细胞术(FCM),检测小鼠T淋巴细胞的表达的活化抗原CD69、CD25、CD71的表达情况;以羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFDA-SE)染色,以FCM分析FS对淋巴细胞体外增殖的影响.结果:终浓度为40、80、160 mg/L的FS均对ConA刺激诱导的T细胞CD69、CD25和CD71的表达有降低作用(P＜0.05).CFDA-SE染色分析显示,上述浓度的FS对ConA诱导的小鼠T淋巴细胞增殖具有抑制作用(P＜0.05).结论:FS对ConA诱导的T细胞早、中、后期活化和体外增殖有抑制作用.     

Investigations on the Age-Dependence of T-Lymphocyte Subpopulations and Lymphocyte Reactivity

The percentage T-lymphocyte subpopulations were determined with the aid of radioactively-labelled monoclonal antibodies to the antigens of total T-lymphocytes and to suppressor T-lymphocyte surface antigens and the responsiveness of the lymphocytes in the presence of ConA and PHA was investigated in groups of 8 subjects aged either between 20 and 30 years or over 75 years. It was found that there was no difference with respect to the response of the lymphocytes to the plant mitogens ConA and PHA. The percentage of suppressor T-lymphocytes, on the other hand, was significantly reduced in the older subjects, whilst the percentage of total T-lymphocytes did not differ significantly from the percentage in younger subjects. These results are discussed in connection with published data.     

Antigen-induced suppression of the in vitro lymphocyte response to different antigens and mitogens

Certain concentrations of antigen stimulated DNA synthesis in sensitized human lymphocytes cultivated in vitro, higher and lower concentrations being less stimulatory. The simultaneous addition of two antigens in low concentrations to the same cells caused an additive response. The decreased response to a high antigen dose did not affect the capacity of the cells to respond to the simultaneous addition of another antigen, as determined at the population level as well as at the cellular level by autoradiography. Presumably specific immunological paralysis was induced by high antigen doses.

Addition of low antigen doses for 1–3 days to human sensitized lymphocytes cultivated in vitro resulted in decreased DNA synthesis as a response to the same antigen added in an optimal dose. Suppression of DNA synthesis was not caused by induction of tolerance or antibody suppression, because the cells also failed to respond to an unrelated antigen and to non-specific mitogens, such as PHA and ALS. Most likely the suppressed response after antigen pretreatment represents a phenomenon analogous to antigenic competition, although this term is not appropriate, since there need not be competition between antigens for a detectable effect. No soluble mediators of suppression could be demonstrated in the supernatant of suppressed cultures.

     

A comparative study of T and B lymphocytes in rainbow trout (Oncorhynchus mykiss) following their separation by nylon wool adherence and lectin agglutination techniques

A nylon wool separation technique was employed to separate rainbow trout leucocytes into adherent and non-adherent populations. The non-adherent population showed a greater response to concanavalin A (ConA) and a lesser response to lipopolysaccharide (LPS) than did the adherent population in the spleen, kidney and peripheral blood. The great majority (>90%) of thymocytes were in the non-adherent population. The non-adherent population from the spleen, kidney and peripheral blood showed signicantly (P>0.05) higher numbers of acid phosphatase-positive lymphocytes than the adherent population, but there was no significant difference in the pattern of immunochemical staining using a mouse anti-trout IgM monoclonal antibody.Soybean agglutinin (SBA) was also employed as a leukoagglutinating reagent to study trout leucocytes. The recovered cells were in two main populations of agglutinated and unagglutinated cells. There was no significant difference in the response of the agglutinated or unagglutinated cells to the mitogens ConA or LPS, or in the staining patterns obtained using acid phosphatase or mouse anti-trout IgM monoclonal antibody.     

Antigen and Unspecific Mitogen Stimulation of Lymphocytes Eluted from Rheumatoid Inflammatory Tissue

Lymphocytes were eluted from synovial tissues of 17 patients with classical rheumatoid arthritis, using a procedure previously reported. Stimulation was obtained wit h the unspecific mitogens phytohemaggalutinin (PHA), pokeweed mitogen (PWM), and concanavalin A (Con A) as well as with purified protein derivative of tuberculin (PPD) and mitomycin-C-treated allogeneic lymphocytes, whereas Candida antigen usually gave a low response. The pattern of reactivity o t unspecific mitogens was similar to that obtained with lymphocytes from peripheral blood of rheumatoid arthritis patients. Two different PPD preparations usually gave transformation of the same magnitude as seen with PHA. This was in contrast to the reactivity of the peripheral blood lymphocytes. It could be demonstrated that the elution procedure initiated some degree of lymphocyte transformation, mainly potentiating the responses to PHA and Con A.     

Depressed T-cell proliferation associated with susceptibility to experimental Taenia crassiceps infection.

Peritoneal infection with Taenia crassiceps cysticerci of naturally resistant (C57BL/10J and C57BL/6J) and susceptible (BALB/cAnN) mice induces a cellular immune depression. T-cell proliferation in response to concanavalin A (ConA) or anti-CD3 was significantly depressed in infected mice of all strains tested. However, in resistant mice, the diminished response to ConA was transient and animals recovered normal responsiveness at day 40, whereas susceptible mice remained suppressed throughout the 40 days of the experiment. In contrast, the proliferative response to anti-CD3 was lower in infected mice than in noninfected controls regardless of differences in natural susceptibility of the strains. Intraperitoneal injection of mice with a parasite extract also induced a depression of the response to ConA, although not as strong as that produced by the parasite itself. This depression is not due to direct effects by parasite antigens over host lymphocytes, as proliferation is not affected by the presence of cysticercal antigens added in vitro. Diminished interleukin-2 production during the parasitosis accounts at least in part for the diminished responses to ConA. A primary infection favors parasite establishment after a second challenge, pointing to the relevance of the immunodepression in generating a host environment favorable to the parasite.     

Decline in mitogen induced proliferation of lymphocytes with increasing age.

We previously reported that mitogen-induced proliferative responses of lymphocytes in the elderly were significantly lower than in young individuals. To determine when this decline occurs, we evaluated the responses of 26-30 subjects of each decile from the third to the tenth decile to the T cell mitogens, phytohaemagglutinin (PHA) and concanavalin A (ConA), and the T dependent B cell mitogen, pokeweed mitogen (PWM). There was a significant decrease in the responses of the 70-, 80- and 90-year-olds to PHA and ConA (less than 40% of the 20-year-olds; P less than 0.01). The 80- and 90-year-olds also showed a decreased response to PWM (approximately 50%; P less than 0.01). The 60-year-olds showed a decreased response to all three mitogens but only the PHA and ConA responses were significantly decreased (P less than 0.01). The 50-year-olds showed a decreased response to ConA, while the 40-year-olds showed decreased responses to both PHA and ConA; both significant at P less than 0.01. The decreased response of the 40-year-olds, however, was only seen in the females. This may be due to the hormonal changes associated with menopause. The general trend of the data suggests that there is a gradual decrease in mitogen-induced proliferative responses with increasing age, large differences become apparent at the age of 60, with a further decrease in the 70s, and most importantly, they remain fairly constant thereafter. Of interest is that only three of the 111 subjects less than 60 years old failed to mount a proliferative response and in each case this was to only one mitogen, while 42 of 118 subjects greater than 60 years old did not respond to at least one mitogen. Ten of these older subjects (2/28 of the 60-year-olds) did not respond to any of the three mitogens (P less than 0.01). This lack of response may be important since we have found a significant association between the lack of response to all three mitogens and increased mortality.     

Correlation between cell-mediated immunity and degree of infection in subjects living in an endemic area of schistosomiasis

Cell-mediated immunity to Schistosoma mansoni antigens, unrelated antigens and mitogens was evaluated in 50 subjects with the same degree of exposure to infection living in an endemic area of schistosomiasis. The degree of infection, assessed by the number of eggs/g of stool, was variable in this population (0-5604), suggesting differences in susceptibility to infection. Absence of lymphoproliferative response was observed in 56% of this group, despite having a response to purified protein derivative of tuberculin (PPD) and tetanus toxoid (TT) antigens and to pokeweed mitogen. The 50 subjects were divided into two groups, according to their degree of infection. The lymphoproliferative responses to schistosomula and adult worm antigens in the group with a low degree of infection (≤400 eggs/g of stool) were higher than the ones documented in patients with a high degree of infection (>400 eggs/g of stool), and these differences were statistically significant (p ≤0.001). An inverse correlation between the lymphocyte proliferation in response to S. mansoni antigens and the degree of infection was also observed (p = 0.02), indicating that subjects with a lower degree of infection have a higher lymphoproliferative response to schistosomula and adult worm antigens. No differences in the lymphocyte reactivity to other antigens (PPD and TT) were detected in these groups. An impairment of interferon-γ in vitro production was observed when the lymphocytes from these subjects were stimulated with S. mansoni adult worm antigen, although they produced gamma interferon in response to phytohemag-glutinin.     

Immunology of streptokinase in human subjects.

Antigens of Plasmodium falciparum, in supernatants of in vitro cultures of the parasite were affinity purified on columns prepared with the IgG fraction of the serum of an immune individual. The purified antigens induced proliferation of lymphocytes from persons who had recently had malaria. The responses were strongest with lymphocytes from individuals infected with falciparum and ovale malaria; vivax malaria infections induced a lower level of response and lymphocytes of unsensitized individuals were little affected. Lymphocytes from unsensitized individuals did not respond to the affinity purified antigen preparations from malaria patients' sera indicating that significant amounts of non-specific mitogens were not present.     

Differences between lymphoid cell populations of guinea pigs and mice as determined by the response to mitogens in vitro.

The stimulation of guinea pig lymphocytes by phytohemagglutinin (PHA), concanavalin A (ConA), methanol-extracted residues of tubercle bacilli (MER), purified protein derivative of tubercle bacilli (PPD), dextran sulphate (DS) and E. coli lipopolysaccharide (LPS), was determined and compared with that of mouse lymphoid cells. The sources of lymphocytes tested were spleen, thymus, lymph nodes and bone marrow. The degree of activation of DNA synthesis by PHA and ConA was higher in guinea pig thymocytes and lymph node cells than in corresponding sources of mouse lymphocytes. The optimum degree of stimulation by PHA and ConA was approximately the same in guinea pig thymocytes, while ConA was by far a better stimulator than PHA for mouse thymocytes. All four B-cell mitogens tested (MER, DS, PPD and LPS) activated DNA synthesis in mouse lymphoid cells while only MER and DS were effective in guinea pig lymphocytes. A guinea pig spleen cell population depleted from B cells was not stimulated, neither by DS nor by MER, while it still responded to PHA and ConA. These results indicate that the proliferative response due to MER and DS occurs in the B-cell compartment. It is suggested that the differences between guinea pigs and mice with respect to their ability to develop a cell-mediated type immunity and to respond to T-independent antigens are related to differences in the relative proportions and degrees of maturation of T- and B-cell subpopulations, as reflected by the selective responsiveness to various mitogens.     

Genetic Control of the In Vitro Responses of Rat Blood Lymphocytes

Blood lymphocytes from the inbred rat strains AS and BN differ in the magnitude both of their in vitro proliferative response to different mitogens and of their in vivo antibody response to the mitogenic fraction of phytohemagglutinin (PHA). We have examined the segregation of in vitro responsiveness to PHA in (AS × BN)F₁× BN backcross rats and have tried to correlate it with other characters that vary in backcross rats. In vitro responsiveness regulated by one or a few loci, is linked to the in vitro responsiveness to B lymphocyte mitogens and the in vivo antibody response to the mitogenic fraction of PHA, but is not linked to the major histocompatibility locus (Ag-B) nor to the frequency of short-lived, small Ig-negative lymphocytes in blood. Lymphocytes from high-responder rats have a shorter lag period before the onset of DNA synthesis in vitro than low-responder rats, and possibly also a higher number of in vitro responding cells. To explain our findings, that the same gene og genes regulate in vitro responsiveness to different mitogens and in vivo antibody response to the mitogenic fraction of PHA, we suggest that the gene or genes act in an immunologically unspecific manner on regulation of lymphocyte proliferation in vitro as well as in vivo.     

Enhancement of in vitro lymphocyte response by neuraminidase

In vitro blastogenic response of sensitized human lymphocytes to specific antigens was significantly enhanced by Vibrio cholerae neuraminidase treatment. No such enhancement was noted when the unsensitized lymphocytes were treated with this enzyme. An enhancement effect of neuraminidase on the lymphocyte response to concanavalin A or pokeweed mitogen (weak mitogens) was also noted; the enzyme had no effect on phytohaemagglutinin (strong mitogen)-induced blasto-genesis. The increased reactivity of lymphocytes is probably related to changed physical and functional properties of the cells after treatment with neuraminidase.     

The appearance of non-specific antibody-forming cells in the efferent lymph draining antigen-stimulated single lymph nodes.

Immunization of single lymph nodes with various antigens led to the appearance of cells in the efferent lymph that secreted antibody specific for the antigen which induced their formation and for a number of unrelated, non-crossreacting antigens. Immunization of single lymph nodes with mitogens led to the appearance of cells secreting antibodies specific for an even greater number of antigens, including one (TNP) that in all probability is not present in the animals' natural environment. When the node was primed with one antigen, a subsequent challenge with an unrelated antigen 12 weeks later led to the appearance of greater numbers of cells containing and secreting antibody against the previously experienced antigen, than was the case in unprimed lymph nodes. These findings indicate that the immune response to antigen provokes the maturation of lymphocytes of specificities unrelated to that of the injected immunogen. Such a mechanism may be important in maintaining immunological memory. Mitogens may directly activate lymphocytes into maturation and expression as antibody-secreting cells, whereas antigens appear to act indirectly.