Autologous mixes lymphocyte reaction in human cord blood lymphocytes: decreased generation of helper and cytotoxic T-cell functions and increased proliferative response and induction of suppressor T cells

T lymphocytes from neonates proliferated significantly more than peripheral blood T lymphocytes from adults in autologous mixed lymphocyte reactions (AMLR). AMLR-activated cord, as compared to adult T lymphocytes, exerted significantly less nonspecific cytotoxic activity on PHA-stimulated adult mononuclear cells and Epstein-Barr virus-transformed target cells. The impaired generation of cytotoxicity of cord T cells was not corrected by Interleukin-2. Blood T lymphocytes from adults activated in AMLR synthesized a helper factor that supported PWM-induced proliferation and immunoglobulin production in both adult and cord B lymphocytes. In contrast, cord blood T lymphocytes failed to produce the helper factor for B lymphocytes. T cells from AMLR cultures established with neonatal lymphocytes showed suppressor activity, as assessed in PWM-stimulated immunoglobulin synthesis of adult peripheral-blood mononuclear cells, significantly higher than that exhibited by T cells from AMLR cultures performed with lymphocytes from adults. Finally, neonatal B lymphocytes could be activated to the production of IgM but not IgG by either adult AMLR-derived helper factor plus PWM or by Epstein-Barr virus, whereas adult B cells secreted both IgM and IgG under the same type of stimulation.     

Cellular cooperation in mitogen-induced human leukocyte inhibitory factor (LIF) synthesis.

Interactions between human T and B lymphocytes and between lymphocyte subpopulations and accessory cells in lymphokine synthesis were investigated. The cells were stimulated with leukoagglutinin (LA), concanavalin A (Con A), protein A (prot A) and anti-β₂-microglobulin (anti-β₂m). The presence of leukocyte inhibitory factor (LIF) in the culture supernatants was tested by the agarose-migration method. The results indicated that monocytes augmented LIF synthesis of T cells but suppressed that of B cells. Monocyte-helper effect was mediated by both cell-cell contact and soluble factors. In addition, T lymphocytes were found to augment B-cell LIF production. B lymphocytes enhanced Con A- but suppressed LA-induced LIF production by T cells. T-cell/B-cell collaboration was based on a direct cell-cell contact and no soluble factors were found.     

Interleukin 1 activity in normal human urine

Human leukocyte dialysates contain components capable of amplifying cutaneous delayed-type hypersensitivity (DTH) reactions. In the present study, two such amplifiers, both less than 3500 m.w., were partially purified from human leukocyte dialysates by gel filtration on Sephadex G-10 followed by high pressure reverse-phase liquid chromatography. These amplifiers of DTH were examined for their effects on production of the migration inhibitory lymphokines leukocyte migration inhibition factor (LIF) and macrophage migration inhibition factor (MIF). The amplifiers were found to increase LIF and MIF production by antigen- or alloantigen-stimulated human peripheral blood lymphocytes in a dose-dependent fashion. Further analysis demonstrated that although antigen-stimulated T4 and T8 cell subpopulations could produce LIF activity under the assay conditions employed, amplification of lymphokine production by modulator was only observed with the T4 subset.     

Elaboration of leukocyte migration inhibitory factor by human lymphocyte subpopulations stimulated with mitogens.

This paper describes experiments to determine whether human lymphocyte sub-populations stimulated with a variety of mitogens, leucoagglutinin (LA), concanavalin A (con A), pokeweed mitogen (PWM), protein A (prot A), and anti-β₂-microglobulin (anti-β₂m), synthesize lymphokines. T and B lymphocytes as well as unseparated mononuclear cells were stimulated with the mitogens, and the presence of leukocyte inhibitory factor (LIF) in the culture supernatants was tested by an agarose migration method. Culture supernatants stimulated with LA or prot A were also fractionated on Sephadex G-100 columns, and LIF-containing fractions were tested for heat stability and the effect of monosaccharides. The results indicated that LA and con A caused LIF synthesis only in T-cell populations, while PWM stimulated both T and B lymphocytes and prot A and anti-β₂mm were B-cell stimulants. Furthermore, LIF from LA-and prot-A-stimulated cultures behaved similarly upon physicochemical characterization.     

Activation of Fc receptor-bearing lymphocytes by immune complexes. I. Stimulation of lymphokine production by nonadherent human peripheral blood lymphocytes

Activation of human peripheral blood lymphocytes by incubation with particulate immune complexes or aggregated human gamma-globulin was studied by measuring the release of leukocyte migration inhibitory factor (LIF) activity. LIF-active supernatants were consistently produced when nonadherent lymphocytes containing less than 1% surface immunoglobulin-bearing cells and less than 0.2% nonspecific esterase-positive monocytes were incubated in the presence of RBC sensitized with rabbit or human antibodies or with pooled heat-aggregated human gamma-globulin. This immune complex-induced lymphokine production (ICLP) was dependent on the presence of cells bearing receptors for the Fc portion of IgG (Fc gamma). ICLP could not be demonstrated with lymphocyte preparations enriched for B cells even though the latter showed vigorous LIF production in the presence of complement-sensitized erythrocytes. ICLP was dependent on the concentration of lymphocytes and of stimulant as well as on the duration of coincubation, and it required active metabolic processes and RNA and protein synthesis but not DNA synthesis. Ca++ but not Mg++ was obligatory. ICLP by non-B Fc gamma receptor-bearing lymphocytes may play a role in antibody-dependent protective inflammation and immunologic injury phenomena, which is similar to that of lymphokine release by antigen-activated T cells in delayed hypersensitivity responses.     

Delineation of suppressor and helper activity within the OKT4-defined T lymphocyte subset in human newborns

Lymphocytes taken from the cord blood of newborns have active suppressor activity. Using in vitro PWM-stimulated cocultures, unfractionated T cells from newborns potently suppressed the expected immunoglobulin G (IgG) synthesis of their mothers' peripheral blood lymphocytes (PBL). Using positive and negative selection techniques, we characterized the active suppressor cell as expressing the OKT4+T8- phenotype. This cord blood lymphocyte subset suppressed maternal IgG synthesis after depletion of maternal suppressor cells, implicating the ability of newborn T cells to suppress directly rather than by inducing adult suppressor activity. Sublethal amounts (1500 rad) of gamma-irradiation fully abrogated the suppressor activity of cord blood T lymphocytes. Radioresistant cord T cells provided T cell help. Irradiation of cord OKT4+ and OKT8+ populations and their subsequent culture with maternal B cells determined that helper activity was a radioresistant subpopulation of the OKT4+ subset. These results indicate significant differences in the functional properties of T cell subsets from adults and newborns. Population studies determined that cord blood lymphocytes had a greater proportion of OKT4+ cells and lower proportion of OKT8+ cells than PBL from unrelated adults. The mothers tested had similar proportions of OKT4+ cells as their babies, and these levels are significantly higher than those of unrelated adults.     

Activation of human lymphocyte subpopulations by protein A from Staphylococcus aureus bacteria.

Cowan I strain Staphylococcus aureus bacteria were found to be mitogenic for human peripheral and cord blood lymphocytes. Experiments with lymphocyte supopulations otained by nylon wool filtration and/or E-rosette separation revealed that T-lymphocytes are the main target cells, whereas isolated B cells did not respond significantly. Further experiments suggested that B cells could be activated in the presence of mitomycin-treated T cells. Null cell-enriched lymphocyte suspensions could be stimulated by Con A but not by the bacteria or by PHA.     

Group C adenovirus DNA sequences in human lymphoid cells.

Human peripheral blood lymphocytes from healthy adults, cord blood lymphocytes, and lymphoblastoid cell lines were screened by hybridization for the presence of group C adenovirus DNA sequences. In 13 of 17 peripheral blood lymphocyte samples from adults, 1 of 10 cord blood samples, and seven of seven lymphoblastoid cell lines tested, results were positive for Group C adenovirus DNA (adenovirus 1 [Ad1], Ad2, Ad5, or Ad6). About 1 to 2% of the lymphocytes carried 50 to 100 viral genome copies per positive cell, as estimated by in situ hybridization. Infectious virus representing all members of group C were recovered, but cultivation in the presence of adenovirus antibody did not cure the cells of free viral genomes. Viral DNA was found in B, T, and N cells but only in 1 of 10 cord blood samples. The results suggest that group C adenovirus infections in childhood result in the persistence of the viral genome in circulating lymphocytes.     

Suppression of adult B cell differentiation in pokeweed mitogen-stimulated cultures by Fc(IgG) receptor-negative T cells from cord blood.

Unfractionated T lymphocytes from cord blood suppressed adult B cell differentiation into immunoglobulin-producing cells in pokeweed mitogen-stimulated co-culture system. Cord blood T cells were fractionated into T cells bearing Fc receptors for IgG (Tgamma cells) and T cells lacking Fc receptors for IgG(Tnon-gamma cells) by rosette formation with ox erythrocytes coated by the IgG fraction of rabbit antisera followed by Ficoll-Hypaque gradient sedimentation. T gamma cells from cord blood, even though isolated after the interaction with immune complexes, showed no suppressor activity on adult B cell differentiation, whereas Tnon-gamma cells exerted strong suppression to a similar extent to that by unfractionated cord T cells. The suppressor activity on B cell differentiation by Tnon-gamma cell as well as by unfractioned T cells from cord blood was completely abrogated by irradiation with 2000 rads. These results indicated that, contrary to suppressor function found in adult T cells, the suppressor activity in cord T cells might be exerted by a T cell subset lacking Fc receptors for IgG(Tnon-gamma cells).     

Regulation of lymphocyte motility by macrophages: characterization of a lymphocyte migration inhibitory factor derived from a macrophage-like cell line

An inhibitory factor on lymphocyte migration was detected using a capillary random migration assay in the culture supernatant of peritoneal exudate macrophages cultured at concentrations greater than 8 x 10(6) cells/ml. After examining different macrophage-like cell lines, J774A.1 cells were found to produce this inhibitory factor, which was termed lymphocyte migration inhibitory factor (LMIF). The inhibitory effect of LMIF on the migration of spleen lymphocytes, thymocytes, and bone marrow cells was determined. The migration of thymocytes was more sensitive to LMIF than was the migration of spleen lymphocytes and bone marrow cells. Interestingly, when the effect of LMIF was tested on the migration of spleen T cells and B cells, T cells were more sensitive than B cells. When the thymocytes were separated by peanut agglutinin into mature and immature thymocytes, the migration of mature thymocytes was more sensitive than that of immature thymocytes, the migration of mature thymocytes was more sensitive than that of immature thymocytes to the effect of LMIF, suggesting that the greatest effect of LMIF was on the migration of mature T cells. Partial purification of LMIF by ion-exchange and gel-filtration chromatography revealed that it is approximately 14,000 in molecular weight and could exist in either monomeric or dimeric forms. The possible role of this factor in an immune response is discussed.     

Effects of labour and medication on major lymphocyte subsets in cord

It is envisaged that flow cytometric analysis of lymphocyte subsets in cord blood may be used as a biomarker for effects on the immune system of exposure to environmental factors. In order to investigate the possible application of this parameter, we first studied the effects of other factors that may influence the outcome of subset analysis in cord blood. FACS analysis was performed in 112 pairs of umbilical cord blood and of peripheral maternal blood sampled at labour. Whereas in maternal blood no statistically significant effects of medication during labour on T lymphocyte numbers and NK cells were found, in oxytocin and in oxytocin and prostaglandin treated mothers B cell numbers showed a statistically significant increase. In cord blood, the course of labour and or medication during labour were identified as the most important factors determining distribution of major lymphocyte subsets. In cord blood after deliveries without medication or after neuroplegic analgesia NPA, the mean percentage of cord blood T lymphocytes CD3 was highest 59 and that of NK lymphocytes CD3- CD16 56 lowest 20 . The mean percentage of T lymphocytes was significantly lower 52 and that of NK lymphocytes higher 28 in cord blood where deliveries were done under NPA in combination with infusion of oxytocin. The combination of NPA with oxytocin and induction of labour by prostaglandin E2 led to a further reduction of T lymphocytes and an increase of NK cells 39 and 38 respectively. The changes in ratio of T and NK lymphocytes were due both to decreasing absolute counts of T lymphocytes and increasing counts of NK lymphocytes. Thus, the effects of labour and or medication during labour must be taken into account when this parameter is applied as a potential biomarker of effects of environmental factors on the immune system.     

Effects of labour and medication on major lymphocyte subsets in cord

It is envisaged that flow cytometric analysis of lymphocyte subsets in cord blood may be used as a biomarker for effects on the immune system of exposure to environmental factors. In order to investigate the possible application of this parameter, we first studied the effects of other factors that may influence the outcome of subset analysis in cord blood. FACS analysis was performed in 112 pairs of umbilical cord blood and of peripheral maternal blood sampled at labour. Whereas in maternal blood no statistically significant effects of medication during labour on T lymphocyte numbers and NK cells were found, in oxytocin and in oxytocin and prostaglandin treated mothers B cell numbers showed a statistically significant increase. In cord blood, the course of labour and or medication during labour were identified as the most important factors determining distribution of major lymphocyte subsets. In cord blood after deliveries without medication or after neuroplegic analgesia NPA , the mean percentage of cord blood T lymphocytes CD3 was highest 59 and that of NK lymphocytes CD3- CD16 56 lowest 20 . The mean percentage of T lymphocytes was significantly lower 52 and that of NK lymphocytes higher 28 in cord blood where deliveries were done under NPA in combination with infusion of oxytocin. The combination of NPA with oxytocin and induction of labour by prostaglandin E2 led to a further reduction of T lymphocytes and an increase of NK cells 39 and 38 respectively . The changes in ratio of T and NK lymphocytes were due both to decreasing absolute counts of T lymphocytes and increasing counts of NK lymphocytes. Thus, the effects of labour and or medication during labour must be taken into account when this parameter is applied as a potential biomarker of effects of environmental factors on the immune system.     

Immune islet killing mechanisms associated with insulin-dependent diabetes: in vitro expression of cellular and antibody-mediated islet cell cytotoxicity in humans

In previous work we have shown that some bacteria can bind to human lymphocytes and can be used to identify lymphocyte subpopulations in conventionally stained blood smears. These bacteria are of different species or genera, which makes it difficult to study the binding mechanism. Also, the main marker for B cells, Brucella melitensis, is of very small size and highly pathogenic. Here we show that B cells as well as some of the T cell subpopulations can be identified by different mutants obtained from a strain of an Escherichia coli. Two procedures were used to generate mutants. First, E. coli-YS57 (pro-his-trp-) was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and the binding to mouse spleen cells was used as a selective pressure. Second, phage-resistant mutants of E. coli-YS57 were obtained and tested for the ability to bind to lymphocytes. Out of 10 strains selected by the former procedure, 5 bound to a significant number of human lymphocytes. All four phage-resistant mutants bound to human lymphocytes. Out of the total of nine mutants that bound to lymphocytes, six bound consistently, i.e., they bound to similar percentages of peripheral blood lymphocytes from different normal donors. One phage-resistant mutant, E. coli USC-106, bound only to B cells. The subpopulations of lymphocytes identified by the mutants were essentially the same as those identified by different species or genera of bacteria. We concluded that E. coli mutants can be obtained that identify human lymphocyte subpopulations and that one of these mutants recognizes B cells; these mutants may be used to study the nature of the receptors for bacteria on lymphocytes, which appear to have a lectin-like nature.     

Structure and biological function of human IgD: XV. Biological potential of IgD-bearing cells from cord blood,and normal human adults

Specifically purified intact IgG anti-human δ or F(ab′)₂ anti-δ stimulated the in vitro incorporation of [³H]thymidine by cord blood and adult human peripheral blood lymphocytes. The magnitude of stimulation was unrelated to the increased frequency of IgD-bearing cells observed in cord blood over that seen in adult blood. Purified T cells lacked the capacity to undergo mitogenesis when incubated with anti-δ antibody. Of special interest was the lack of anti-δ enhancement of phytohemagglutinin (PHA) responsiveness of cord blood lymphocytes that is often observed when adult peripheral blood lymphocytes are treated with anti-δ, then PHA. This observation could be interpreted as indicating: (a) that the anti-δ-activated neonatal B cells, or their lymphokines, were preferentially activating a population of suppressor cells, which in turn prevented the PHA-augmentation usually seen with adult cells, (b) that the neonatal B cells do not possess the T cell-augmenting capacity or, (c) that the neonate lacks a subpopulation of T cells that can be acted upon by the B-cell or B-cell factors.     

Adherence of bacteria to human lymphocyte subpopulation and the role of monosaccharides in bacterial binding

In the present investigation lymphocyte bacterial binding, receptors for bacteria on lymphocytes, and the relationships between bacterial binding and lymphocyte activation were studied. Of the strains used Escherichia coli, Bacillus subtilis, and Corynebacterium xerosis bound to both purified T and B cells. Staphylococcus aureus, Staphylococcus albus, and Brucella melitensis bound chiefly to B lymphocytes. Monosaccharides and treatment of lymphocytes with lectins, enzymes, or sodium metaperiodate affected bacterial adherence. Thus the lymphocyte receptors for bacteria appeared to contain carbohydrate moieties. There was no clear-cut correlation between lymphocyte binding of bacteria and bacterium-induced leukocyte-inhibitory factor (LIF) synthesis and proliferation.     

Identification of tissue transglutaminase as a novel molecule involved in human CD8+ T cell transendothelial migration

During inflammation, T lymphocytes migrate out of the blood across the vascular endothelium in a multistep process. The receptors mediating T cell adhesion to endothelium are well characterized; however, the molecules involved in T cell transendothelial migration (TEM) subsequent to lymphocyte adhesion to the endothelium are less clear. To identify receptors mediating TEM, mAbs were produced against human blood T cells adhering to IFN-gamma-activated HUVEC in mice and tested for inhibition of lymphocyte TEM across cytokine-activated HUVEC. Most of the mAbs were against beta(1) and beta(2) integrins, but one mAb, 6B9, significantly inhibited T cell TEM across IFN-gamma, TNF-alpha, and IFN-gamma plus TNF-alpha-stimulated HUVEC, and did not react with an integrin. 6B9 mAb did not inhibit T cell adhesion to HUVEC, suggesting that 6B9 blocked a novel pathway in T cell TEM. The 6B9 Ag was 80 kDa on SDS-PAGE, and was expressed by both blood leukocytes and HUVEC. Immunoaffinity purification and mass spectrometry identified this Ag as tissue transglutaminase (tTG), a molecule not known to mediate T cell TEM. Treatment of HUVEC with 6B9 was more effective than treatment of T cells. 6B9 blockade selectively inhibited CD4(-), but not CD4(+), T cell TEM, suggesting a role for tTG in recruitment of CD8(+) T lymphocytes. Thus, 6B9 is a new blocking mAb to human tTG, which demonstrates that tTG may have a novel role in mediating CD8(+) T cell migration across cytokine-activated endothelium and infiltration of tissues during inflammation.     

Amplification of the activity of human leukocyte inhibitory factor (LIF) by the generation of a low molecular weight inhibitor of PMN leukocyte chemotaxis

Leukocyte inhibitory factor (LIF), which was derived from human peripheral blood lymphocytes by stimulation with concanavalin A ad partially purified by Sephadex G-100 gel filtration, inhibited the in vitro spontaneous migration and chemotaxis of human PMN leukocytes as assessed in a Boyden chamber micropore filter assay. The inhibitory activity was attributed to LIF, a principle defined in terms of its inhibition of PMN leukocyte migration from glass capillary tubes since it was preferentially directed to PMN leukocytes as compared to mononuclear leukocytes, exhibited a size comparable to LIF by gel filtration, and was inactivated by diisopropyl fluorophosphate in parallel with LIF. Incubation of PMN leukocytes with LIF released additional inhibitory activity, distinct from LIF, which resembled the neutrophil-immobilizing factor (NIF) by virtue of its approximate m.w. of 4000 by filtration on Sephadex G-25, inactivation by trypsin digestion, and preferential noncytotoxic inhibition of spontaneous migration and chemotaxis of PMN leukocytes as compared to mononuclear leukocytes. Thus LIF inhibits PMN leukocyte migration both by a direct action on the cells and by an amplification pathway that is mediated by low m.w. chemotactic inhibitors similar to NIF.     

Improvement of decreased interleukin 2 (IL-2) responsiveness and IL-2 production in autologous mixed-lymphocyte reaction of cord blood lymphocytes by interferon-gamma and IL-2 and the role of HLA-DQ antigen

Cord blood T cells did not produce interleukin 2 (IL-2) nor acquire responsiveness to it in autologous mixed-lymphocyte reaction (AMLR) as they do when activated by phytohemagglutinin (PHA). The ability of the cells to respond to IL-2 was restored either by the addition of recombinant IL-2 to the AMLR culture or by the preculture of non-T stimulator cells with recombinant interferon-gamma (IFN-gamma). IL-2 production was also induced when the T cells were added with recombinant IL-2 at the initiation of the AMLR culture, preceded by the treatment of non-T cells with recombinant IFN-gamma. IL-2-producing cells of cord blood induced in the above-mentioned condition were defined to be OKT4+ T cells, because the deletion of OKT4+ T cells from T-cell population abrogated the reaction, while that of OKT8+ T cells did not. Acquisition of IL-2 responsiveness and IL-2 production of T cells seemed to be mediated by HLA-DR and HLA-DQ molecules of non-T cells because these reactions were blocked by the treatment of non-T cells either with monoclonal anti-HLA-DR or with anti-HLA-DQ antibody. The HLA-DR and HLA-DQ densities of cord blood non-T cells were low as compared with those of adult, but the expression of HLA-DQ was remarkably improved by IFN-gamma treatment. In regard to IL-2, both IFN-gamma and IL-2 were needed to enable the lymphocytes to produce. This may suggest that some functional maturation by IL-2 of responder T cells is further required. These combined data suggested that cord blood non-T cells are defective as a stimulator in AMLR and this could be corrected by enhancing the expression of HLA-DQ antigen.     

Alterations in the human immune response to the hepatitis B vaccine among the elderly

The specific binding of hepatitis B (HBs) antigen by lymphocytes from old people immunized with hepatitis B vaccine was explored. For that purpose HBs antigen was combined with fluorescent microspheres, and labeled antigen was allowed to react with lymphocytes from HBs vaccine-responsive or unresponsive people. Lymphocytes from 10 responders and 14 nonresponders were tested for their antigen-binding ability. For controls, lymphocytes were incubated with microspheres bearing human albumin. Lymphocytes from 8 out of 10 responders were able to recognize HBs antigen; for the nonresponders the ratio was 9 out of 14. HBs-binding lymphocytes were B cells but not T lymphocytes. B and T cells from responders and nonresponders were combined and cultivated for 8 days in the presence of HBs antigen, and antibody-producing cells were counted. Neither B cells alone nor B cells plus T cells from nonresponders were able to produce antibody. On the other hand B cells from unresponsive old people produced antibodies when they were cultivated in the presence of HBs antigen and T cells from responsive old people. These data suggest that some elderly individuals who do not produce antibody after in vivo immunization by HBs vaccine do have antibody-producing cells. Instead of a gap in their immune repertoire, these people are suffering from immune dysfunction.     

Electrophoretic mobility of human lymphocytes purified by nylon columns and spontaneous rosettes]

Cell electrophoresis enables the separation of the lymphocytes in normal human blood into two principal groups, as a function of their migration speed in relation to 1 mum.sec -1..v-1.cm. In 42 healthy adults, 19,9 % of the lymphocytes have a slower migration, and 80,1 % a faster migration than the reference speed. Two known methods are used for the selection of the lymphocytic populations : spontaneous rosetting with sheep red blood cells, a property of the T lymphocytes, and the adherence to nylon wool columns, which is dominant in the case of B lymphocytes. The cells which do not form spontaneous rosettes, and the cells adhering to nylon wool columns have above all a slow migration. On the contrary, cells which do not adhere to nylon columns have a fast migration. These arguments are in favour of the T nature of the rapid migrating lymphocytes, and of the B nature of the slow migrating lymphocytes.