Basic types and vital forms of human peripheral and PHA-stimulated lymphocytes studied in vitro

Natural diversity in peripheral and PHA-stimulated lymphocytes seen in the same donors was studied using digitized streak photo of living cells in observational camera. Cells were monitored for 5-8 h at the superior limit of optical resolution by means of phase-contrast microscopy. Intact lymphocytes were observed in autological blood plasma, and PHA-stimulated lymphocytes were examined in self-conditioned centrifuged growth medium. The majority of intact cells were small- and middle-sized floating lymphocytes with microvilli, and middle-sized caudate lymphocytes capable of stick-slip motion. The lesser part consisted of "spread-eagle" or movable forms of both large granular lymphocytes and middle-sized lymphocytes of several types: narrow-plasm lymphocytes with lamellipodia, wide-plasm lymphocytes without cytoplasmic processes, lymphocytes with single pseudopodia, and lymphocytes with single lobopodia of complex shape. On the contrary, the minor fraction of PHA-stimulated lymphocytes of 3 day old cultures contained floating cells with microvilli or floating cells with microvilli and two pseudopodia, whereas the majority of these lymphocytes were spread-eagle or movable forms of cells of different type. These substrate-adhesive PHA-stimulated lymphocytes had well defined apical and basal cell surfaces, but upon mechanical stress are easily pinched off to become ball-shaped. At least 6 different cell types were distinguished among substrate-adhesive PHA-stimulated lymphocytes, with more than half of these being heavily vacuolated spheroid lymphocytes prone to forming cell clusters. The rest PHA-stimulated lymphocytes were represented by signet-ring lymphocytes with dark or light cytoplasm, narrow-plasm lymphocytes with large prolonged nuclei and lamellipodia, lymphocytes with single lobopodia, and lymphocytes with single spiral structures in the cytoplasm. The spiral structure is 10-11 microns in length and 0.5-0.7 micron in width, being presumably a mitochondrion or a group of butt-joined mitochondria. Since some of the caudate middle-sized lymphocytes also contain this structure, these may be regarded as putative precursors of respective type of PHA-stimulated lymphocytes. Under the conditions of observation, interphase nuclei of all live PHA-stimulated lymphocytes were seen to contain numerous globular or fiber structures of condensed chromatin made of 0.3-0.8 micron beads. These beads are doubtless interphase chromomeres.     

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.     

Concentration of nucleotides and deoxynucleotides in peripheral and phytohemagglutinin-stimulated mammalian lymphocytes. Effects of adenosine and deoxyadenosine

Concentrations of purine and pyrimidine ribonucleotides were measured with HPLC in lymphocytes of man, horse, pig and sheep and in rat thymocytes. The ATP concentration was highest in lymphocytes of all species and about 850 pmol/10(6) cells in human and equine lymphocytes, higher in porcine and lower in ovine lymphocytes and rat thymocytes. The GTP concentration was comparable in human, equine and porcine lymphocytes, but lower in ovine lymphocytes. ATP concentration was also measured in lymphocytes of man, horse and pig with a luciferin-luciferase assay. During culturing with or without phytohemagglutinin the ATP concentrations decreased in these lymphocytes. The concentrations of TTP and dATP were measured with a DNA polymerase assay. Phytohemagglutinin-stimulation increased the TTP concentration in lymphocytes of all three species, the dATP concentration only in human lymphocytes. ATP, TTP and dATP concentrations and thymidine incorporation were measured in phytohemagglutinin-stimulated lymphocytes after 24 and 48 h culturing in the presence of adenosine or deoxyadenosine. Adenosine increased the ATP concentration in porcine and equine, but not in human lymphocytes. Deoxyadenosine and adenosine did not affect the TTP concentration. Deoxyadenosine decreased the ATP concentration only in the presence of EHNA in human lymphocytes, but increased it in other conditions and in equine and porcine lymphocytes. Deoxyadenosine in the presence of EHNA increased the dATP concentration in human, equine and porcine lymphocytes 3-, 10-, and 9-fold, respectively, and decreased considerably thymidine incorporation. Deoxyadenosine without EHNA increased the dATP concentration 2-5-fold, decreased the thymidine incorporation in lymphocytes of man and horse, but stimulated incorporation in porcine lymphocytes about 5-fold. The latter results indicate that accumulation of dATP is not always associated with inhibition of cell proliferation.     

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.     

The control of autoreactivity. I. Lack of autoreactivity in murine spleens is due to concomitant presence of suppressor and autocytotoxic lymphocytes.

To determine if autocytotoxic lymphocytes were naturally occurring in murine spleens, C57BL/6 spleen cells were fractionated on discontinuous BSA gradients. Autocytotoxicity was assessed in vitro (cytotoxicity of C57BL/6 fibroblasts) and in vivo (splenomegaly and popliteal node enlargement in C57BL/6 mice). A medium density subpopulation of lymphocytes was shown to be autocytotoxic and to be similar to autoreactive lymphocytes produced by the in vitro "sensitization" of splenic lymphocytes on syngeneic fibroblasts monolayers. The naturally occurring autocytotoxic lymphocytes express no detectable theta antigen, did not adhere to nylon, but did have an Fc receptor. In recombination experiments, BSA-fractionated lymphocytes were incubated with autocytotoxic and "sensitized" lymphocytes. A light density subpopulation was shown to suppress both autoreactive lymphocyte subpopulations at a 1:50 ratio. The suppressor cells were nylon nonadherent T lymphocytes. The lack of autoreactivity of unfractionated murine spleen cells is due to the concomitant presence of autocytotoxic and suppressor lymphocytes. If suppressor lymphocytes are selectively removed in vitro, the reactivity of autocytotoxic lymphocytes can be detected.     

Regulation of lymphocyte production in the bone marrow. I. Turnover of small lymphocytes in mice depleted of B lymphocytes by treatment with anti-IgM antibodies

To examine the concept that the genesis of lymphocytes in the bone marrow may be regulated by homeostatic feedback signals from peripheral B lymphocytes or their products, lymphocyte production was measured in mice selectively depleted of B lymphocytes by repeated administration of anti-IgM antibodies from birth. The turnover of small lymphocytes was quantitated radioautographically after DNA labeling by continuous infusion of 3H-thymidine. In the femoral marrow of anti-IgM-treated mice, the number of small lymphocytes was reduced and their turnover time was shorter than in control mice, presumably reflecting the premature elimination from the marrow of maturing cells about to express surface IgM. The absolute number of small lymphocytes being produced per femur in unit time, however, was identical in anti-IgM-treated and control mice. Lymphocyte production in the thymus was also unaffected by anti-IgM suppression whereas in the spleen the turnover of small lymphocytes was reduced due to the lack of young immigrant B lymphocytes from the bone marrow. The results demonstrate that the normal large-scale production of lymphocytes in mouse bone marrow is independent of the magnitude of the peripheral pool of B lymphocytes or the level of circulating immunoglobulins, suggesting the process is not subject to feedback control. Some implications for the genesis and diversity of primary B lymphocytes are discussed.     

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.     

Glycosphingolipids of purified human lymphocytes.

Biochemical analysis of the glycosphingolipids (GSLs) of human lymphocytes revealed qualitative and quantitative variations among purified lymphocytes from different tissues. The major neutral GSLs of tonsil lymphocytes are glucosyl ceramide (CMH), lactosyl ceramide (CDH), trihexosyl ceramide (CTH), and globoside. Thymocytes and peripheral blood lymphocytes (PBL) contain only traces of CTH and globoside, and PBL contain more CMH and CDH per cell than tonsil lymphocytes. Thymocytes and PBL contain relatively large amounts of more complex neutral GSLs that are present in only trace amounts in tonsil lymphocytes. Peripheral blood lymphocytes contained three and five times more lipid-bound sialic acid than thymocytes and toncil lymphocytes, respectively. Thymocytes and PBL contained mostly hematoside, whereas tonsil lymphocytes contained more complex gangliosides in addition to hematoside. The observed differences in GSL content among these cells may be related to their content of B cells, which comprise approximately 50% of tonsil lymphocytes, 10% of PBL and 0-2% of thymus cells, and/or the known differences in functional capacities of cells in different lymphoid organs. These findings suggest that cell surface GSLs may serve as markers for identification of functional subpopulations of human lymphocytes.     

Multi-functionality of allospecific cytotoxic T-lymphocytes in vitro

The data presented demonstrate the capacity of allospecific cytotoxic lymphocytes to fulfill noncytolytic regulatory functions. Cytotoxic lymphocytes suppress cell proliferation induced during development of a response in reactions of a mixed culture of lymphocytes and blast transformation irrespective of haplotypes of the cells involved in these reactions. At the same time, cytotoxic lymphocytes do not practically affect spontaneous proliferation of T-cells. The inhibitory effect of cytotoxic lymphocytes is expressed only upon direct contact with activated T-lymphocytes and is not relate to their apoptosis and death. The results obtained suggest polyfunctionality of allospecific cytotoxic lymphocytes expressed in the capacity of these lymphocytes to fulfill both effector (target lysis) and regulatory functions.     

Polyfunctionality of Allospecific Cytotoxic T-Lymphocytes in in vitro Systems

The data presented demonstrate the capacity of allospecific cytotoxic lymphocytes to fulfill noncytolytic regulatory functions. Cytotoxic lymphocytes suppress cell proliferation induced during development of a response in reactions of a mixed culture of lymphocytes and blast transformation irrespective of haplotypes of the cells involved in these reactions. At the same time, cytotoxic lymphocytes do not practically affect spontaneous proliferation of T-cells. The inhibitory effect of cytotoxic lymphocytes is expressed only upon direct contact with activated T-lymphocytes and is not relate to their apoptosis and death. The results obtained suggest polyfunctionality of allospecific cytotoxic lymphocytes expressed in the capacity of these lymphocytes to fulfill both effector (target lysis) and regulatory functions.     

Preliminary investigation of the characteristics of fish lymphocytes separated on a Percoll discontinuous gradient

This investigation was carried out to determine if lymphocyte functional heterogeneity could be demonstrated in teleosts on the basis of density difference. The lymphocytes separated into two fractions on a Percoll discontinuous gradient. The less dense lymphocytes separated at 1.056gml⁻¹ and contained both moderately villous and smooth lymphocytes, whereas the more dense lymphocytes separated at 1.07 g ml⁻¹ and were predominantly moderately villous lymphocytes. The moderately villous lymphocytes had a higher mitochondria content than the smooth lymphocytes. Lymphocytes separating in the 1.056g ml⁻¹ layer were more responsive to stimulation with polyclonal mitogen (PHA) than the lymphocytes separating in the 1.07 g ml⁻¹ layer. This indicates that the lymphocytes separated according to either cell lineage or maturity.     

In vitro induction of suppressor cells by plasma of rats with Trypanosoma brucei rhodesiense infection

Mitogenic responses of B and T lymphocytes from spleens of rats infected with Trypanosoma brucei rhodesiense were suppressed. Plasma from infected rats suppressed the mitogenic responses of B and T lymphocytes from spleens of normal uninfected rats. Removal of immune complexes from plasma of infected rats significantly reduced the suppressive effect of the plasma on splenic lymphocytes of normal uninfected rats. Normal thymus cells treated with plasma from infected rats and added to cultures of normal spleen lymphocytes inhibited the mitogenic responses of B and T lymphocytes. We suggest that the interaction of immune complexes and Fc or C3b receptors of T lymphocytes resulted in the in vitro induction or activation of T suppressor lymphocytes.     

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.     

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.     

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.     

Activity of acid phosphatase and succinate and dehydroorotate dehydrogenases during the interaction of allogeneic lymphocytes and target cells in tissue culture]

A study was made of the activity of acid phosphatase in interaction of lymphocytes and target cells; it was shown that in the lymphocytes its greatest increase occurred in the 1- and sometimes in the 3-hour cultures; sometimes the activity of the enzyme in the L-cells increased by the 3rd-6th hour. The initial activity of acid phosphatase was greater in the immune lymphocytes; later, on their addition to the L-cell culture, there was no significant difference in the changes in the activity of the enzyme in the immune and normal lymphocytes. In the L-cells the activity of acid phosphatase was lower on addition of normal lymphocytes in comparison with the immune ones. The activity of dehydrogenases in the lymphocytes increased by the 3rd hour of the incubation period and was greater in the immune lymphocytes than in the normal ones. The activity of the succininc- and dehydroorotic dehydrogenases altered practically at the same periods in the L-cells as in the lymphocytes. The intensification of the activity of the redox enzymes and of the acid phosphatase during the first hours of contact pointed to the rapid activation of the effector lymphocytes.     

Scanning electron microscopy of homing and recirculating lymphocyte populations

The surface structure of T and B lymphocytes in vivo was investigated using scanning electron microscopy. For these studies the spleen and mesenteric lymph node of mice enriched for B lymphocytes (adult thymectomized, lethally irradiated, bone marrow reconstituted mice, B mice) and of mice enriched for T lymphocytes (adult, lethally irradiated, thymocyte transferred mice, T mice) were examined. Both types of lymphocytes demonstrated a smooth cell surface when they were situated in their respective microenvironment, whereas recirculating T and B cells exhibited numerous microvilli on the cell surface. In postcapillary venules, known to be the major sites of entry of lymphocytes in lymph nodes, lymphocytes were in contact with the endothelial wall by means of these microvilli. While passing the endothelial lining, lymphocytes withdrew their microvilli and appeared smooth upon arrival in the lymphatic stroma. It is suggested that microvilli on the surface of lymphocytes play a role in cellular recognition mechanisms.     

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.     

Preferential Binding of Mouse Mammary Tumor Virus to B Lymphocytes

Mouse mammary tumor virus (MMTV) has been shown to preferentially infect B lymphocytes in vivo. We have used recombinant envelope-coated fluospheres and highly purified MMTV particles to study the distribution of the viral receptors on fresh mouse lymphocytes. A preferential dose-dependent binding to B lymphocytes was observed which could be competed with neutralizing antibodies. In contrast, T-lymphocyte binding remained at background levels. These results strongly suggest a higher density of viral receptor molecules on B lymphocytes than on T lymphocytes and correlate with the preferential initial infection of B lymphocytes observed in vivo.     

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.