Antigen-specific suppressor T lymphocytes (Leu-2a+3a-) in human schistosomiasis japonica

Immunoregulatory mechanisms in human schistosomiasis japonica were investigated through in vitro T lymphocyte-macrophage co-culture experiments. High responder T lymphocytes responded to the schistosomal adult worm antigen in the presence of HLA-DR-compatible low responder macrophages, whereas low responder T lymphocytes failed to respond to this antigen even in the presence of HLA-DR-compatible high responder macrophages. This observation strongly suggested that T lymphocytes, but not macrophages, controlled the low responsiveness to the schistosomal adult worm antigen. When high responder lymphocytes were challenged with the schistosomal antigen in the presence of low responder T lymphocytes, low responder T lymphocytes caused an antigen-specific suppression, in a dose-dependent manner, of the responsiveness of high responder lymphocytes. The suppressor T lymphocytes were mitomycin C resistant and were positive for Leu-2a, but were negative for Leu-3a. When Leu-2a+ T lymphocytes were depleted from low responder T lymphocytes, a vigorous response by the Leu-2a-depleted T lymphocytes (Leu-2a-3a+) to the schistosomal adult worm antigen was observed. By adding back the Leu-2a+ T lymphocytes into autologous T lymphocyte subpopulations, the specific response to the adult worm antigen was completely suppressed. These findings clearly demonstrate the existence of Leu-2a+3a- suppressor T lymphocytes that can control low responsiveness to the schistosomal adult worm antigen in man.     

Direct suppression of lymphocyte induction by the immunoregulatory human serum low density lipoprotein, LDL-In

Preparations of LDL-In, an immunosuppressive lipoprotein subfraction, were analyzed for the capacity to directly suppress the response of human lymphocytes to the representative stimulant PHA vis-a-vis indirect mechanisms mediated by soluble factors or cell:cell interactions. Serum lipoprotein subfraction enriched in LDL-In induced a suppressed state in lymphocytes during 18-hr induction cultures. These lymphocytes, whether partially or completely suppressed, when added to fresh responder lymphocytes in the presence of PHA did not suppress the response of the responder lymphocytes. In contrast, the major low density lipoprotein (LDL) did not suppress lymphocytes at equivalent concentration in the induction culture, nor did LDL-exposed lymphocytes suppress responder lymphocytes. The supernatant medium from LDL-In-suppressed lymphocytes did not contain a newly synthesized or released suppressive factor. Finally, LDL-In-suppressed lymphocytes were not rescued by normal lymphocytes. Each of these observations, and previous evidence that adherent cells do not mediate the biologic effects of LDL-In, support the hypothesis that the biologic manifestations of LDL-In suppression of lymphocyte function result from a direct effect on the lymphocyte that is exposed to this lipoprotein, possibly via the previously demonstrated LDL-In receptor.     

Lymphocytes binding and T cell mitogenic properties of group A streptococcal lipoteichoic acid.

We previously reported that lipoteichoic acid (LTA) of group A streptococci binds spontaneously to mammaliam cell membranes via lipid moieties ester-linked to the LTA molecule. We now describe biochemical and immunologic evidence that LTA binds to human and murine lymphocytes as an early event in the induction of mitogenesis in T lymphocytes. The biochemical studies showed that binding of radiolabeled LTA to lymphocytes was lymphocyte-concentration, and temperature dependent, and it reached a maximum in 15 min. Binding was reversible and specific with a dissociation constant of 89 micrometer for adult lymphocytes and 57 micrometer for cord blood lymphocytes. Immunologic studies showed that the LTA was mitogenic only for T lymphocytes. Dose response curves of lymphocyte mitogenesis induced by LTA and the binding of LTA to intact lymphocytes were shown to be related. The results suggest that LTA binds to specific receptor sites on T lymphocytes to trigger the mitogenic response.     

Application of orthogonal light scattering for routine screening of lymphocyte samples

Orthogonal and forward light-scattering properties of lymphocytes were measured from patients with different lymphocytic diseases in order to determine the potential value of light scattering as a screening device. Monitoring of orthogonal light scattering of lymphocytes of a B-cell chronic lymphocytic leukemia patient during splenic irradiation (SI) revealed the selective decrease of malignant cells and the fact that the major part of the residual lymphocytes were cytotoxic lymphocytes. By combining forward and orthogonal light scattering it was shown that lymphocytes from a patient with T gamma lymphocytosis were abnormal. Orthogonal light scattering also showed an increase in cytotoxic lymphocytes in a patient with mononucleosis infectiosa and in a splenectomized patient. Orthogonal light scattering of lymphocyte subpopulations showed that the leu8+ population of a patient with mononucleosis infectiosa was bidisperse. For elderly donors the occurrence of CD3+, CD4+, CD8+, and HNK-1+ lymphocytes with a large orthogonal light scattering varied considerably. The CD8+ lymphocytes of these donors consisted mainly of cytotoxic lymphocytes. These results show that determination of light-scattering properties of lymphocytes may yield important diagnostic information and can indicate when further investigation of the lymphocytes by means of immunofluorescence is necessary.     

Quantitative study of deoxycytidine incorporation in large and small lymphocytes of the mouse

Using radioautographic smear preparations of thymocytes and mesenteric lymph node (MLN) cells labelled with three different tritiated pyrimidine deoxyribonucleosides, the incorporation of DNA precursors was studied separately on large lymphocytes and small lymphocytes. Radioautographic reaction due to generally tritiated deoxycytidine ( [G-3H]CdR) labelling in vivo in large lymphocytes was more intense than that in small lymphocytes. When mice were sacrificed 6 hr after the administration of tritiated thymidine ( [3H]TdR), small lymphocytes were labelled more heavily than large lymphocytes. However, labelling intensity with [3H]TdR in large lymphocytes was greatly enhanced by the administration of 5-fluoro-deoxyuridine, whereas in small lymphocytes labelling intensity was only fairly enhanced by the same treatment. When cells were incubated in vitro with 5-tritium labelled deoxycytidine [( 5-3H]CdR) for 10 min, there was no significant difference in labelling intensities between large and small lymphocytes. In the case of [G-3H]CdR incorporation, the labelling intensity in large lymphocytes was found to be significantly stronger than that in small lymphocytes. Large as well as small lymphocytes incorporated [3H]TdR very well in vitro. However, addition of 5 X 0 X 10(-5) M of non-radioactive CdR to the medium greatly decreased the incorporation of [3H]TdR by large lymphocytes, whereas the effect of non-radioactive CdR in small lymphocytes was not so marked as that in large lymphocytes. Furthermore, the [3H]TdR-labelling percentages were decreased at the same rate by the addition of non-radioactive CdR in both large and small lymphocytes. These results indicate that large lymphocytes and a proportion of small lymphocytes have a strong tendency to convert CdR to thymidine mono-phosphate, which is utilized for DNA synthesis, whereas this ability is relatively weak in the rest of small lymphocytes. Thus, it is probably that this metabolic ability changes during the transition of the large lymphocyte to the small lymphocyte.     

Quantitative parameters of the IgG binding by Fc receptors and the functional activity of blood lymphocytes in healthy cattle and cattle with chronic lympholeukemia

The binding of IgG to lymphocyte Fc receptors in the blood of healthy cattle and of cattle with chronic lympholeukemia has been studied by fluorometric techniques before and after the incubation of lymphocytes in a serum-free medium at 37 degrees C. The study has shown that changes in the intensity of binding of IgG to Fc receptors of normal and leukemic lymphocytes correlate with changes in the cell-mediated cytotoxic activity of the corresponding lymphocytes. Lower cell-mediated cytotoxic activity of leukemic lymphocytes in comparison with that of normal lymphocytes was parallelled by a lower association constant of IgG with leukemic lymphocytes Fc receptors.     

Morphological Differences between Thymus- and Bone Marrow-Derived Lymphocytes

Simple morphological criteria are described, enabling one to distinguish two types of lymphocytes in normal unstimulated mice: 'Th', predominant in the thymus (90-92%) and 'Bm', predominant in the bone-marrow (85-93%). Their distribution in the peripheral lymphoid organs agreed significantly with the observed average distribution of T and B lymphocytes. Thy-mectomized and irradiated bone-marrow-restored CBA mice, as well as nude mice, showed a striking lack of Th cells. In vitro depletion of θ-bearing or Ig-bearing lymphocytes lead to a correlated depletion of Th or Bm lymphocytes, respectively: blood lymphocytes treated with anti-θ serum + complement showed a decrease in Th lymphocytes corresponding to the percentage of cells killed by the anti-θ serum. The selective retention of Ig-bearing spleen cells on Wigzell's anti-lg columns caused a depletion of Bm lymphocytes. It is concluded that the Th type is the morphological expression of the T lymphocytes, and the Bm type that of the B lymphocytes. The implications of this way of distinguishing T and B lymphocytes by simple morphology, and of the actual relationships between T and B cell differentiation, are discussed.     

The entry of T and B lymphocytes into rat popliteal lymph nodes undergoing a graft-versus-host reaction

The entry of radiolabeled blood-borne T and B lymphocytes into resting popliteal lymph nodes and popliteal lymph nodes stimulated with semiallogeneic lymphocytes was investigated in rats. Thoracic duct lymphocytes separated into T- and B-lymphocyte populations on nylon-wool columns were radiolabeled with 51chromium and equal numbers of T or B lymphocytes were injected intravenously. While the ratio of T and B lymphocytes in the blood is approximately 3:1 it was found that the ratio of T to B lymphocytes migrating into lymph nodes was approximately 9 T to 1 B lymphocyte in both resting and antigenically stimulated lymph nodes. Since the ratio of T to B lymphocytes in thoracic duct lymph is similar to that of blood, there is a disparity between the number of T cells entering and leaving lymph nodes. These results suggest that some T lymphocytes may return to the blood directly and/or there is increased T lymphocyte death in lymph nodes.     

Characteristics of the cyclic nucleotide phosphodiesterases of normal and leukemic lymphocytes

The specific activity of cylic AMP phosphodiesterase and cyclic GMP phosphodiesterase of leukemic lymphocytes was 5–10-fold greater than that of purified normal lymphocytes or homogenates of spleen, thymus or lymph nodes of normal mice. This rise was demonstrable over a wide range of substrate concentrations. Both normal and leukemic lymphocytes contained a heat-stable, calcium-dependent activator of phosphodiesterase. However, the increased activity of phosphodiesterase in leukemic lymphocytes was not due to this protein activator since (a) phophodiesterase activity from these cells was not stimulated by this activator and (b) phosphodiesterase activity of leukemic lymphocytes was not inhibited by the calcium chelator, ethyleneglycol-bis-(β-aminoethylether)-N′,N′-tetraacetic acid, suggesting that the enzyme was not already maximally activated. A comparison of several other properties of phosphodiesterase from normal and leukemic lymphocytes showed that the enzymes have similar pH optima, similar stabilitis to freezing and thawing and similar sensitivities to inhibition by the phosphodiesterase inhibitors, chlorpromazine, papaverine and isobutylmethylxanthine. However, the subcellular distribution of the phosphodiesterases was different, and the phosphodiesterase of leukemic lymphocytes was significantly more resistant to heat than that of normal lymphocytes.Although no differences were found between the phosphodiesterases of normal and leukemic lymphocytes in their sensitivities to drugs, there were marked differences in drug sensitivity between the phosphodiesterase of lymphocytes and that of other tissue. For example, concentrations of chlorpromazine which inhibited phosphodiesterase of cerebrum by 70% had no effect on phosphodiesterase activity of lymphocytes. On the other hand, the papaverine-induced inhibition of phosphodiesterase was similar in lymphocytes and cerebrum.Since an optimal concentration of cyclic nucleotides is essential to maintain normal cell growth, these results suggest that the abnormal growth characteristics of leukemic lymphocytes may be explained by their high activity of phosphodiesterase. Furthermore, the qualitative and quantitative differences between the phosphodiesterases of leukemic lymphocytes and other tissues raise the possibility of selectively inhibiting the phosphodiesterase of the leukemic lymphocytes, thereby reducing their rate of growth, without affecting other tissues.     

Normal T-cell receptors for alloantigens

To study the diversity of normal mouse T lymphocytes capable of mediating allograft immunity, we modified a cell culture system so that both induction of sensitization and target cell damage could be studied in vitro. Mouse lymph node lymphocytes were sensitized in vitro against allogeneic fibroblasts. The sensitized lymphocytes produced immunospecific cytotoxic effects against target fibroblasts in vitro. We found that T lymphocytes were directly involved in both sensitization and cytotoxicity.We used this allograft system to separate nonsensitized mouse lymphocytes on the basis of their ability to bind to allogeneic fibroblasts. Adhering lymphocytes were found to be enriched in effector cells following sensitization. The nonadhering lymphocytes showed a decreased ability to undergo sensitization against fibroblasts that were syngeneic to the ones used for adsorption. However, they were able to become sensitized against unrelated fibroblasts of another H-2 phenotype.These findings indicate that specific receptors for histocompatibility antigens pre-exist on diverse populations of normal mouse T lymphocytes.     

Ribonuclease deficiency in lymphocytes of patients with chronic lymphocytic leukemia (CLL)

Ribonuclease (RNase) activity in the lymphocytes of 20 chronic lymphocytic leukemia (CLL) patients and 10 normal subjects was studied. It was found that in the lymphocytes of the control subjects the RNase activity could be detected in the pH range 4.5 to 8.6, inclusive. The RNase activity versus pH profile of normal lymphocytes consists of an acid RNase peak at pH 6.5 and alkaline RNase peak at pH 7.8. When treated with pCMB an inhibitor-bound RNase activity was revealed. The peak of this activity lay between pH 6.7 to 7.0. Liberating the inhibitor-bound RNase activity changed the RNase activity-pH profile, yielding one peak curve with a maximum at pH 7.0. RNase activity in CLL lymphocytes was remarkably lower than that in normal lymphocytes. The acid RNase in 80% of the CLL patients was lower by a factor of ten. Likewise, a many fold decrease in alkaline RNase activity (in some cases down to the zero level) was observed in CLL lymphocytes. However, in 70% of CLL patients, a level of the inhibitor-bound RNase activity was similar to that found in normal lymphocytes. In 20% of the studied CLL patients, a remarkable decrease in both free alkaline and inhibitor-bound RNase activity was observed. When poly-C was used as a substrate for determining RNase activity, a decrease to approximately 15% in CLL lymphocytes was observed, when poly-U was used instead of poly-C, a decrease to 65% was found only as compared with normal lymphocytes. This may suggest that CLL lymphocytes are deficient in a poly-C specific RNase which displays its activity within a neutral and alkaline pH range.     

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. VI. Roles of T and B lymphocytes in cluster formation.

The role of immune T and B lymphocytes in the in vitro production of antigen-specific clusters with macrophages pulsed with soluble protein antigen was studied by assaying the cluster-producing capability of lymphocyte populations deprived of either T or B lymphocytes. Populations deprived of B lymphocytes were able to produce clusters to the same extent as unfractionated lymphocyte populations, whereas populations deprived of T lymphocytes produced very few clusters. Staining of the cluster lymphocytes for membrane Ig using the immunoperoxidase technique showed that Ig-bearing cells were to a large extent excluded from the clusters. We suggest that each cluster is initiated by an antigen-committed T lymphocyte and that the assay for clusters may be used to enumerate the number of antigen-committed T lymphocytes in a given population.     

Interactions between human lymphocytes and paramyxovirus-infected cells: adsorption and cytotoxicity.

The capacity of human lymphocytes to adhere to paramyxovirus-infected monolayers and their capacity to kill paramyxovirus-infected cells was investigated. A large fraction of human lymphocytes was found to adhere firmly to the paramyxovirus-infected monolayers. Predsorption of lymphocytes on mumps virus-infected cells impaired their adsorption to a second cell monolayer of the same type. The cytotoxic activity of lymphocytes against mumps virus-infected cells was also reduced after predsorption on mumps virus- or Newcastle disease virus-infected (NDV) cell monolayers. Exposure of lymphocytes to trypsin did not significantly decrease either adsorption or cytotoxicity. Pretreatment of lymphocytes with neuraminidase (NANase) partly inhibited adsorption whereas cytotoxicity was not decreased. Cell fractionation experiments after rosetting of the lymphocytes with sheep erythrocytes (E) indicated that T cells were equally or better adsorbed than "non-T" cells. Taken together with previous experiments which showed that the majority of T lymphocytes are not cytotoxic against mumps virus-infected cells these results suggest that adherence of lymphocytes to infected cells and cytotoxicity may be unrelated phenomena.     

Decidual lymphocytes of human spontaneous abortions induce apoptosis but not necrosis in JEG-3 extravillous trophoblast cells

The human decidua contains an unusually high proportion of lymphocytes, mainly NK and T cells, which are potentially cytotoxic to the trophoblast when they are stimulated with certain cytokines. Given the high incidence of spontaneous abortion in humans and other species, our working hypothesis is that decidual lymphocytes are involved in immunological mechanisms that attack the trophoblast and induce abortion when any gestational problem arises. To test this hypothesis, flow cytometry was used to compare decidual lymphocyte populations in first-trimester spontaneous abortions and elective terminations of first-trimester pregnancy. We found significantly higher proportions of decidual lymphocytes that expressed activation markers, and of T cells (mainly T helper cells) in spontaneous abortions than in elective terminations of pregnancy. Decidual lymphocytes from spontaneous abortion, like decidual lymphocytes from elective termination of pregnancy and peripheral blood lymphocytes, were however, unable to lyse the JEG-3 extravillous cytotrophoblast cell line in a (51)Cr-release assay. Nevertheless, decidual lymphocytes from spontaneous abortion, unlike decidual lymphocytes from elective termination of pregnancy and peripheral blood lymphocytes, induced apoptosis in JEG-3 cells as determined by DNA fragment-release assay. Hematoxylin and eosin staining showed a significantly higher proportion of apoptotic JEG-3 cells when these cells were treated with decidual lymphocytes from spontaneous abortion than when JEG-3 cells were cultured with decidual lymphocytes from elective termination of pregnancy. The ultrastructural signs of apoptosis were confirmed by electron microscopy. These data support the hypothesis that activated decidual lymphocytes participate in human spontaneous abortion by inducing apoptosis but not necrosis of the trophoblast.     

Murine isolated lymphoid follicles contain follicular B lymphocytes with a mucosal phenotype

Isolated lymphoid follicles (ILFs) are organized intestinal lymphoid structures whose formation can be induced by luminal stimuli. ILFs have been demonstrated to act as inductive sites for the generation of immune responses directed toward luminal stimuli; however, the phenotype of the immune response initiated within ILFs has largely been uninvestigated. To gain a better understanding of the immune responses initiated within ILFs, we examined phenotypic and functional aspects of the largest cellular component of the murine ILF lymphocyte population, B lymphocytes. We observed that murine ILF B lymphocytes are composed of a relatively homogenous population of follicular B-2 B lymphocytes. Consistent with their proximity to multiple stimuli, ILF B lymphocytes displayed a more activated phenotype compared with their counterparts in the spleen and Peyer's patch (PP). ILF B lymphocytes also expressed higher levels of immunomodulatory B7 and CD28 family members B7X and programmed death-1 compared with their counterparts in the spleen and PP. ILF B lymphocytes preferentially differentiate into IgA-producing plasma cells and produce more IL-4 and IL-10 and less interferon-gamma compared with their counterparts in the spleen. Immunoglobulin repertoire analysis from individual ILFs demonstrated that ILFs contain a polyclonal population of B lymphocytes. These findings indicate that murine ILFs contain a polyclonal population of follicular B-2 B lymphocytes with a phenotype similar to PP B lymphocytes and that, in unchallenged animals, ILFs promote immune responses with a homeostatic phenotype.     

Interaction of the lymph node and splenic lymphocytes of mice in inactivation of allogeneic hematopoietic stem cells

A study was made of interaction of mouse spleen and lymph node lymphocytes in inactivation of allogeneic stem cells. It was established that T lymphocytes of the lymph nodes and spleen lymphocytes do not interact on combined administration; their action is of additive nature. B lymphocytes of the lymph nodes have a regulating activity both in respect to T lymphocytes of the lymph nodes and lymphocytes of the spleen. The stem cells serve as target. Depending on the stem cells/B lymphocytes ratio B lymphocytes are capable of exerting either helper or suppressor action.     

cytophotometric,flow cytofluorometric and morphometric demonstration of the existence of an antigen dependent thymus lymphocyte subpopulation

Summary Using absorption cytophotometry and flow cytofluorometrical DNA and protein estimation of single thymus lymphocytes we were able to establish that after injection of a large dose of antigen (ovalbumin) a subpopulation of lymphocytes arises in the thymus with high protein contents above that of those lymphocytes normally present, however, in small quantities in the thymus. By morphometrical analysis it was established that these lymphocytes are situated in the outermost cortex.     

The influence of cell cycle kinetics on the radiosensitivity of Down's syndrome lymphocytes

In agreement with previous work, [60Co]gamma-irradiation shortly after phytohemagglutinin (PHA) stimulation, induces higher frequencies of chromosomal aberrations in trisomy 21 lymphocytes compared to normal controls. However, equal frequencies of chromatid aberrations are induced in fully-stimulated trisomy 21 and normal lymphocytes by irradiation during G2. We have observed that trisomic lymphocytes respond more rapidly to PHA stimulation than normal lymphocytes. Furthermore, we have observed that chromosomal radiosensitivity increases as a function of time after PHA stimulation in normal lymphocytes. When normal lymphocytes are irradiated 8 h after PHA stimulation, the frequencies of chromosomal aberrations induced are comparable to those induced in trisomy 21 lymphocytes irradiated 30 min after PHA stimulation.     

CD5+ B cells predominate in peripheral tissues of rabbit.

Restricted usage of VH genes is observed in rabbit B lymphocytes and in human and murine CD5 B lymphocytes. This observation raised the possibility that most rabbit B lymphocytes were CD5+. To investigate this we cloned the CD5 gene from a rabbit cosmid library, using a probe derived from human CD5 cDNA. The rabbit CD5 gene was transfected into a murine T cell line and then we used the transfectants to develop anti-rabbit CD5 mAb. By Western blot analysis, the mAb reacted with a 67-kDa protein in lysates prepared from mesenteric lymph node and spleen cells. We determined the frequency of CD5+ B lymphocytes in peripheral lymphoid tissues of adult rabbits by two-color immunofluorescence analysis using anti-CD5 mAb and anti-L chain antibodies. The analysis showed that essentially all peripheral B lymphocytes in adult rabbits express CD5. The observation that CD5 is expressed on nearly all rabbit B lymphocytes contrasts markedly to mouse and human, where only a small number of B lymphocytes express CD5. We propose that most peripheral B lymphocytes in rabbit, as in chicken, develop early in ontogeny and are maintained throughout life by a self-renewing process.     

High anti-TNP plaque-forming cell potential of residual mIg+ cells in a T cell population

In the course of experiments designed to study the immune response of purified populations of B lymphocytes to thymus-independent (TI) antigens, a variety of cell purification procedures were followed. In using anti-immunoglobulin-coated dishes to separate lymphocytes bearing membrane immunoglobulin (mIg) from mIg- lymphocytes, it was found that the nonadherent fraction, which was predominantly mIg-, complement receptor negative, and nonresponsive to the B cell mitogen lipopolysaccharide, gave very substantial anti-TNP plaque-forming cell responses to 2 TI antigens. These responses could be inhibited by incubation of such cells in the presence of anti-mu and thus appeared to be attributable to mIg+ cells. The evidence suggests the existence of a population of B lymphocytes that constitute a minor component of mIg+ cells having a high potential to make in vitro antibody responses. Users of techniques that utilize anti-Ig as a tool for separating B and T lymphocytes should carefully assess the extent to which residual B lymphocytes in the mIg- population contribute to antibody responses being studied.