Cytotoxic effector cell function in organized gut-associated lymphoid tissue (GALT).

Lymphocytes from the organized gut-associated lymphoid tissues (GALT) of adult guinea pigs were examined for surface markers characteristic of T and B lymphocytes and for their capacity to function as effector cells in mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) reactions. GALT lymphocytes formed rosettes with rabbit erythrocytes, a T-cell marker, and underwent proliferative responses in vitro in the presence of phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM). GALT lymphocytes were cytotoxic in vitro for erythrocyte and DBA mastocytoma targets in the presence of PHA. A population of GALT lymphocytes bound aggregated γ-globulin; however, they functioned poorly in ADCC reactions. Thus, organized GALT in the guinea pig contains lymphocytes capable of functioning in T-cell-dependent MICC reactions but either lacks the effector cell population which mediates ADCC or contains an effector cell which functions poorly in ADCC.     

Insoluble immune complexes suppress mitogen-induced proliferation of human T lymphocytes bearing Fc gamma receptors.

Human peripheral blood lymphocytes were mixed with erythrocyte-antibody (EA) complexes and separated into EA-rosette forming cell (EA-RFC)-enriched and EA-RFC-depleted suspensions. Thymidine incorporation of EA-RFC-enriched population in the presence of T cell mitogens (PHA, Con A, PWM) was about half of that of EA-RFC-depleted or of unseparated cells. The dose-response curves and kinetics of proliferation were found to be very similar in the three populations. Proliferative response of EA-RFC-enriched lymphocytes was strictly T cell dependent, although non-T cells were later recruited to incorporate thymidine. The interaction of T lymphocytes bearing surface receptors for IgG (T_G) with insoluble complexes followed by a post-binding temperature sensitive event, resulted in the modulation of Fc receptors associated with an impaired proliferative response to PHA, Con A, and PWM, without significant change in metabolic cell activity as shown by cell viability, sponaneous leucine incorporation, or β₂ microglobulin release.     

Separation of mitogen-induced suppressor cells of human antibody-producing cells

Human antibody-forming cells were demonstrated by a plaque in agar technique following in vitro stimulation with either pokeweed mitogen or Cowan I strain of protein A-positive Staphylococcus aureus bacteria. We evaluated the effects on this antibody formation caused by the addition of cells which had been stimulated with PH A or Con A. Both Con A and PHA cells harvested after 3 days showed strong inhibition of pokeweed-induced plaque formation. The majority of the suppression could be accounted for by a blast fraction separated on 1g sedimentation gradients from the Con A or PHA cultures. Small cells from such cultures showed inhibition of PFC when added at high ratios (1:2), but this suppressive activity diluted out much more rapidly than that of the blast cells. No helper activity was noted with either small cells or blasts. Our studies indicate a T-cell blast as the suppressive fraction in Con A- or PHA-stimulated human lymphoid cells. While this T-cell suppression applies to T-dependent responses such as antibody stimulation with pokeweed mitogen, it does not have a substantial effect on Cowan I-induced plaque-forming responses. The finding that Cowan I-induced plaques could not be inhibited by Con A or PHA blasts indicates the T independence of this response.     

Passive transfer of experimental allergic encephalomyelitis in the Lewis rat with activated spleen cells: differential activation with mitogens

It has previously been shown that spleen cell transfer of clinical EAE requires donor cells to be cultured in vitro prior to transfer. Donor cells must be stimulated when cultured, and either Con A or the encephalitogen, guinea pig myelin basic protein (BP), satisfies this stimulation requirement. Following recovery from passive disease, recipients of these in vitro cultured cells will subsequently develop clinical symptoms of EAE sooner than controls when challenged with BP in complete Freund's adjuvant (BP-CFA). In the present study, three T-cell mitogens were evaluated as donor cell stimulants in the required in vitro culture period. Pokeweed mitogen (PWM) as well as Con A stimulated the donor cell population to the degree that clinical EAE could be transferred with 5 × 10⁶ cultured viable cells. Con A at culture levels below 0.25 μg/ml did not yield transfer active cells even though proliferation levels were similar to those found at concentrations of Con A that did yield transfer active cells. Phytohemagglutinin (PHA)-stimulated cultures did not transfer clinical disease even though the degree of lectin induced proliferation ([³H]thymidine uptake as well as recovered cells from culture) was equivalent to the PWM- or Con A-stimulated, transfer positive, cultures. Mixing experiments suggested that the inability of PHA or low doses of Con A to induce transfer active cells was not due to the induction of suppressor cells. Although cells cultured with PHA do not transfer clinical EAE, recipients of these cells as well as recipients of either PWM- or Con A-stimulated donor cells develop an early appearance of disease upon subsequent challenge with BP-CFA. This included cells incubated with a concentration of Con A (0.1 μg/ml) which did not induce cells capable of transferring clinical EAE. These results suggest that PHA and perhaps the low dose of Con A may stimulate the proliferation of the EAE effector cell precursor population without causing the additional differentiation of this precursor population into the effector cell population which is capable of transferring clinical disease. Alternatively, PHA may expand only the helper cell population while effective doses of Con A and PWM would expand both helper and effector cell populations.     

Stimulatory effect of Bestatin,a new specific inhibitor of aminopeptidases,on the blastogenesis of guinea pig lymphocytes

A potent protease-inhibitor of Actinomycetes origin, Bestatin. which is of dipeptide nature and inhibits aminopeptidase B and leucine-aminopeptidase competitively, strongly stimulates blastogenesis of small lymphocytes triggered with polyclonal mitogen. such as phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and lipopolysaccharide of Escherichiae coli (LPS), whereas it inhibits DNA synthesis of normal resting lymphocytes. The stimulatory effect is non-selective with respect to the category of small lymphocytes, i.e. T- and B-lymphocytes, but strikingly selective with respect to the stage of blastogenesis: the stimulation is greatest at a relatively early stage, diminishes as mitogen-activation proceeds, and is not appreciable at a later stage of lymphocyte blastogenesis.The pattern of Bestatin stimulation on lymphocyte blastogenesis is specific for the mitogen used: in T-lymphocyte activation with PHA or Con A, the stimulation first increases and then decreases with increase in mitogen concentrations, whereas in B-lymphocyte activation with LPS, with increasing concentrations of the mitogen, the stimulation increases to a plateau at approximately 100 μg/ml of mitogen. The optimum concentration of Bestatin was found to be approximately 50 μg/ml (0.16 mM) for either PHA or Con A activation, and 50 to 75 μg/ml for B-cell activation with LPS. Bestatin must remain in cultures of T- and B-lymphocytes with polyclonal mitogens for at least about 24 and 16 hr, respectively, to exert its stimulatory effect on blastogenesis.Biochemical results, together with those from autoradiographic analyses, indicate that Bestatin increases the number of blastoid-transformed lymphocytes with polyclonal stimulants. It is suggested that aminopeptidases, possibly located at the cell surface, may play a role in the control of lymphocyte activation during immune responses.     

Phylogeny of lymphocyte heterogeneity. II. Differential effects of temperature on fish T-like and B-like cells.

Lymphocytes from the bluegill, a freshwater fish, were observed to undergo in vitro mitogenic responses to a variety of “classical” mitogens. Using cell fractionation approaches based upon surface markers and in vitro mitogenesis, bluegill lymphocytes could be divided into two populations. One population responded to PHA and Con A but not to LPS, contained surface antigens in common with bluegill brain, and did not form spontaneous rosettes with rabbit erythrocytes. The other population responded to LPS but not to PHA or Con A, did not appear to contain surface antigens in common with brain, and did form rosettes with rabbit erythrocytes. The former population responded to mitogenic stimulation very well at 32 °C, whereas the latter population responded better at 22 than at 32 °C. The pattern of mitogenic responses and brain antigen distribution coupled with the observation that mixed lymphocyte responses were obtained at 32 but not at 22 °C makes it likely that the 32 °C responsive population represents the fish equivalent of T cells. The other population may be B cells. These data suggest that the immunosuppressive effects of low temperatures on cold-blooded animals may be effects on the generation of functional T cells and not on B cells.     

Effect of T- and B-lymphocyte mitogens on interactions between lymphocytes and macrophages.

The mitogenic response of murine T cells ² to Con A, S-Con A and PHA was found to be macrophage-dependent. Optimal mitogenic responses were obtained when macrophage-depleted T-cell populations were reconstituted with 5% normal peritoneal macro-phages. Studies were carried out to investigate the effect of T- and B-cell mitogens on in vitro physical interactions between murine lymphocytes and macrophages. This was done by determining the number of T- or B cells binding to macrophages in the absence and in the presence of T- and B cell mitogens, and comparing the results of these experiments with the induction of lymphocyte proliferation. Con A increased the binding of T cells to macrophages when used in mitogenic doses (1–5 μg/ml). Dose response experiments showed that the same dose of Con A which produced maximal mitogenic stimulation also induced the greatest number of T cells to bind to macrophages. Nonmitogenic doses of Con A (20–50 μg/ml) did not enhance the binding of T cells, while identical doses of S-Con A both induced T cell mitogenesis and increased the number of T cells bound to macrophages. Similar results were obtained with PHA. None of the B-cell mitogens tested (LPS, EPO 127 and LAgl) increased the binding of either T or B cells to macrophages. PWM, which is mitogenic for both T and B cells, increased the binding of T cells to macrophages, but not that of B cells. In brief, the four T-cell mitogens tested (Con A, S-Con A, PHA, and PWM) induced specific physical interactions between T cells and macrophages, while none of the B-cell mitogens had any effect on the physical interactions between either B or T cells and macrophages when used in mitogenic doses.     

Modulation of lymphocyte functions by group A streptococcal membrane

Protoplast membranes isolated from group A streptococci suppress functions of mouse B cells in vivo and in vitro. Intraperitoneal injection 24 or 72 hr (but not 12 hr) before collection of lymphoid cells results in a selective decrease in the mitogenic response of bone marrow cells to dextran sulfate (DS). The response of bone marrow cells to lipopolysaccharide (LPS), and spleen cells to both DS and LPS, is unaltered. In vitro exposure of lymphocytes to membranes concomitantly with mitogen reduces the response to both DS and LPS, however, the DS response is more susceptible to low doses of membrane. Suppression of the response to DS in vitro is not mediated by cells bearing Thy 1.2 antigen. Neither the phytohemagglutinin (PHA)-responsive cells nor the adherent cells participate in suppression of the LPS response in vitro. In contrast to the suppression of B-cell functions neither the PHA nor concanavalin A (Con A) response of mouse bone marrow, spleen, or thymus cells is altered by streptococcal protoplast membranes injected 24 hr before collection of cells. In vitro exposure of spleen cells to a limited range of concentrations of membrane results in an enhanced proliferative response of spleen cells stimulated by suboptimal doses of PHA. This synergism is not mediated by the adherent cells. Addition of membranes to spleen cell cultures in vitro has no effect upon the response of spleen cells to suboptimal doses of Con A or to optimal doses of either Con A or PHA. Higher concentrations of membranes reduce the proliferative response of both control and mitogen-stimulated cells. This nonselective suppression by high doses of membranes is not due to toxicity. Delayed hypersensitivity to sheep erythrocytes is potentiated by injection of membranes. These studies suggest that streptococcal membranes preferentially suppress the immature B cells and enhance certain T-cell functions.     

Selective T cell killing of human lymphocytes by ultraviolet radiation

The effects of ultraviolet radiation (uv) on human B and T lymphocytes were studied. In vitro studies showed that T lymphocytes were more sensitive to uv than B lymphocytes as assessed by eosin-dye exclusion. Following uv exposure, the viable lymphocytes responded to mitogens (PHA, PWM), and functional B lymphocytes were present at a time when no viable T cells were detected. Varying doses of uv were required to abrogate different in vitro responses (proliferative response to antigen or allogeneic cells, MIF production, and cell-mediated lympholysis). In vivo, uv was able to diminish an established cutaneous delayed hypersensitivity response. In vitro uv treatment of parental mouse spleen cells eliminated a graft-versus-host reaction in F₁ recipients as determined by the spleen index. The basis for the differential effect of uv on B and T lymphocyte viability and functional responses is unknown.     

Facilitation of adenylate cyclase stimulation in macrophages by lectins.

Preincubation of guinea pig peritoneal macrophages with concanavalin A (Con A) markedly enhanced the accumulation of 3′,5′-cyclic-adenosine monophosphate (cAMP) in response to the adenylate cyclase (AC) stimulators prostaglandin E₁ (PGE₁) and isoproterenol (IP). Basal cAMP levels were not altered. Maximal enhancement of cAMP accumulation was induced by preincubation with 50–100 μg/ml Con A for 10 min at 37 °C. Con A-induced facilitation of macrophage responsiveness was prevented by α-methyl-d-mannoside (αMM). No facilitation was induced by the divalent derivative, succinyl-Con A or by Con A immobilized on Sepharose beads. Con A-induced facilitation developed normally in macrophages treated with the microfilament blocking agent, cytochalasin B. The responsiveness of macrophages to PGE₁ and IP was also augmented by phytohemagglutinin (PHA) but wheat germ agglutinin (WGA), soy bean agglutinin (SBA), pokeweed mitogen (PWM), and Lotus tetragonolobus lectin (LL) showed no enhancing effect. The effect of Con A on cAMP levels was the result of augmented cAMP synthesis and not of reduced degradation or a block in cAMP egress from the cells. Lectin-induced facilitation of AC stimulation could be mediated via one of the following mechanisms: (i) induction of receptor clustering; (ii) causing a conformational change in the receptors; (iii) inhibition of negative cooperativity; (iv) causing an increase in membrane fluidity; (v) disruption of microtubules by acting as a Ca²⁺ ionophore; or (vi) inactivation of a sugar-containing inhibitor of AC.     

Fluorescence-activated cell sorting of human T and B lymphocytes: I. Direct evidence that lymphocytes with a high density of membrane-bound immunoglobulin are precursors of plasmacytes

Human peripheral lymphocytes bearing either a high or a low amount of membrane-bound immunoglobulin were studied. Cells were “tagged” with fluorescein-labeled antiimmunoglobulin reagents and separated by means of a new electronic instrument, a fluorescence-activated cell sorter (FACS), into populations with either > 10⁵ or < 5 × 10³ immunoglobulin molecules per cell. Fractions of high purities were obtained. (>80% and >99.9%, respectively). In vitro, different functional properties were observed: lymphocytes with high densities of membrane-Ig gave a late proliferative response after stimulation with Pokeweed mitogen (PWM). A considerable proportion of stimulated cells developed into mature plasmacytes as detected by cytoplasmic staining. Those lymphocytes with a low density or complete absence of membrane-Ig could be stimulated by both Phytohemagglutinin (PHA) and Pokeweed mitogen, but no differentiation into plasmacytes occurred. The functions are similar to those of bone marrow-derived (B) and thymus-derived (T) lymphocytes in mice. Thus, the designation as B lymphocytes for human lymphocytes with a large quantity of membrane-bound immunoglobulin seems justified.     

Thymus-dependent and thymus-independent effector functions of mouse lymphoid cells. Comparison of cytotoxicity and primary antibody formation in vitro

We have compared two effector functions, antibody formation and cytotoxic capacity in vitro, of mouse cells of various origin with special reference to the T lymphocyte dependence of these processes. We have used addition of PHA and coating of target chicken erythrocytes (CRBC) with antibody as the two means of inducing cytotoxicity. Antibody formation in vitro has been studied both against thymus-dependent sheep erythrocytes (SRBC) and thymus-independent (E. coli) antigens. Spleen cells from thymectomized, lethally irradiated bone marrow-, or fetal liver-repopulated mice were deprived of phagocytic cells by uptake of colloidal iron. They did perform better than normal spleen cells in the antibody-induced cytotoxicity and were also induced to cytotoxicity by PHA. PHA did not induce increased DNA synthesis in these T cell-deprived spleen cell preparations, which could not make primary antibodies to SRBC but were able to do so against E. coli antigens. Fresh bone marrow and fetal liver cells, deprived of phagocytic cells, were also induced into a highly efficient cytotoxicity by anti-CRBC as well as by PHA. Pretreatment of spleen cells with an alloantiserum (θ) against T lymphocytes reduced but did not abolish the PHA-induced cytotoxicity. In contrast, it did not affect the antibody-induced cytotoxicity. Such treated cells could not make antibodies to SRBC but could do so against E. coli. Pretreatment of spleen cells with a heteroantiserum (MBLA) against mouse B lymphocytes completely abolished all cytotoxic- and antibody-forming abilities of the cells, although experiments with combinations of θ-treated and MBLA-treated cells suggested that the MBLA treatment had left behind a significant portion of helper T cells needed for the in vitro antibody response. From these data we conclude, as have others, that the antibody-induced cytotoxicity is independent of T lymphocytes. It can be induced in immature precursor cells from fetal liver or bone marrow, and these cells may also become cytotoxic on interaction with PHA. However, in normal spleen cells, at least part of the PHA-induced cytotoxicity is T cell dependent. Some preliminary data suggest that this PHA-induced cytotoxicity of normal spleen cells may be a joint process between T lymphocytes and other cells.     

Further characterization of mitogen-induced autorosette-forming cells: correlation with T-cell subsets and with lymphocyte proliferative responses to mitogens

Spontaneous autologous rosette-forming cells (ARFC), which form rosettes with autologous erythrocytes, have been of interest as a subset of thymus-derived lymphocytes (T cells). An association of these cells with concanavalin A (Con A)-induced ARFC has been suggested. Furthermore, the Con A-induced ARFC have been shown to be a suppressor T-cell subset in the Con A-generated suppressor system. We have previously reported the induction of ARFC from T cells by several T-cell mitogens such as phytohemagglutinin-P (PHA) and allogeneic non-T cells other than Con A. In the present report, we further characterized the mitogen-induced ARFC and have extended the study to patients with systemic lupus erythematosus (SLE). We have found that ARFC are also inducible from peripheral blood T cells by pokeweed mitogen (PWM). Studies of T-cell surface markers on the ARFC using OKT monoclonal antibodies confirmed the induction of ARFC from both OKT4- and OKT8-reactive T cells by either Con A, PHA, or PWM stimulation. However, OKT4-reactive T cells were the major cellular source of the ARFC induced by all of the mitogens. In studies of SLE patients, proportions of both Con A- and PWM-induced ARFC were found to be significantly low in PBL of SLE patients treated with moderate or large doses of prednisone, with or without concomitant immunosuppressants, but not in SLE patients without such treatment. Proportional analysis of the T cells and their subsets suggested association of these alterations in the mitogen-induced ARFC with the OKT4-reactive T cells, since a significant decrease in the OKT4-reactive T-cell subset was demonstrated in the PBL of these patients. Proportions of PHA-induced ARFC, however, were not significantly different between SLE patients and healthy adults. Moreover, positive correlations of the mitogen-induced ARFC with lymphocyte proliferative responses to each mitogen were established in both SLE patients and healthy adults. These results further support our previous observation that suggest the receptors for autologous erythrocytes are enhanced or reexpressed on those T cells which are highly activated by mitogens.     

Periodate-induced lymphocyte transformation : II. Character of response and comparison with phytohemagglutinin and pokeweed mitogen stimulation

Sodium metaperiodate is mitogenic for human peripheral lymphocytes. Evidence of stimulation can be detected as increased thymidine incorporation at 72 h after only 10 sec of exposure to the IO₄⁻. The degree of response varies with lymphocytes from different donors, but maximum stimulation for the healthy donors studied was obtained at concentrations of IO₄⁻ between 10⁻³ M and 4 × 10⁻³ M. Concentrations of 8 × 10⁻³ M and above are non-stimulatory and toxic. Exposures to optimum concentrations for 1 h or longer result in essentially no stimulation and inincreased cell death. However, a significant response to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) remains. The kinetics of response over a 4 day culture period are similar for IO₄⁻, PHA and PWM. The morphology of the blast cells and the degree of response suggest that the IO₄⁻ responsive lymphocyte population may be more closely related to the PWM stimulated cells than the PHA responsive lymphocytes.     

Macrophages from human colostrum. Multinucleated giant cell formation by phytohemagglutinin and concanavalin A

Macrophages obtained from human colostrum were cultured in the presence of phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM). PHA caused multinucleated giant cell formation which could be inhibited by the addition of N-acetyl-d-galactosamine. Con A caused multinucleated giant cell formation and was cytotoxic in higher concentrations. Both effects could be inhibited by addition of α-methyl-d-mannoside and α-methyl-d-glucoside. PWM did not cause multinucleated giant cell formation but was cytotoxic in high concentrations.     

The assay, properties, and kinetics of human T cells forming clusters with sheep erythrocytes (CFC) in stimulated cultures.

This study describes a new method of detecting the in vitro stimulation of lymphocytes based on the appearance of cells having the property to cluster several layers of SRBC around themselves (CFC). The formation of multilayer rosettes is temperature dependent and requires trypan-blue-excluding, metabolically active blastoid cells. Non-separated cells as well as purified T cells stimulated with allogeneic leucocytes (MLR), specific antigens, or polyclonal mitogens (PWM, Con A) gave rise to CFC. Multilayer rosettes were not formed by separate B cells. In the MLR, CFC were detected 48 hr after the beginning of cultures and increased in number thereafter just like thymidine incorporation. The response to Con A and PWM was detected earlier and gave rise to two or three peaks, the first rise in the number of CFC coinciding with the peak of thymidine incorporation but the maximum increase occuring two or three days later. Treatment of blastoid cells with a serum specific for T cells, but not with an anti-immunoglobulin serum, abolished their ability to form ordinary or multilayer rosettes. When separated, however, on a nylon wool column, CFC were more adherent than the bulk of T cells. It is suggested that CFC form a subpopulation of T cells with distinct characteristics, allowing a direct assessment of membrane changes which take place when T lymphocytes are activated in vitro.     

Ly-4.2: a cell membrane alloantigen of murine B lymphocytes. III. In vitro studies

The alloantigenic specificity Ly-4.2 is present on a restricted population of murine lymphocytes which have previously been shown to have some of the properties generally ascribed to B lymphocytes, both with regard to distribution and function. In the study reported herein, the effect of anti-Ly-4.2 and anti-Thy-1.2 (θ) antisera have been examined in various in vitro systems. (a) T cell-mediated lysis of ⁵¹Cr-labeled P815-X2 target cells by immune allogeneic peritoneal exudate cells is inhibited by anti-Thy-1.2, but not affected by the anti-B (Ly-4.2) reagent. (b) Antibody-dependent lymphocyte-mediated lysis of ⁵¹Cr-labeled sheep red cells was only slightly inhibited by anti-Ly-4.2 and anti-Ig antisera, and not at all by anti-Thy-1.2 antisera, indicating that this type of cell lysis is mediated by neither T (Thy-l⁺) nor B (Ly-4.2⁺,Ig⁺) cells. (c) The response of lymph node lymphocytes to various mitogens was affected thus: PHA, completely inhibited by anti-Thy-1.2 but not by anti-Ly-4.2; Con A, largely inhibited by anti-Thy-1.2, and slightly by anti-Ly-4.2; PWM (pokeweed mitogen), partially inhibited by both antisera; E. coli endotoxin lipopolysaccharide, greatly inhibited by anti-Ly-4.2 but only slightly by anti-Thy-1.2. The findings demonstrate that anti-Thy-1.2 reacts predominantly with T cells and anti-Ly-4.2 with B cells.     

Immunomodulatory role of thyroid hormones: in vitro effect of tetraiodothyronine (T4) on mitogen induced blastogenic response of lymphocytes

An attempt was made to find out the immunomodulatory role of thyroid hormone, tetraiodothyronine (T4), and its effect on in vitro mitogen induced blastogenesis. Human peripheral blood lymphocytes (PBL) were subjected to phytohaemagglutinin (PHA) concanavalin-A (Con. A) and pokeweed mitogen (PWM) in presence or absence of T4. Basal blastogenic response was significantly enhanced in dose related manner by T4. PHA and Con.A induced response was depressed significantly (r = -0.975 and r = -0.945) whereas less than 50 ng T4 in presence of PHA showed mild stimulation. On the other hand, PWM induced response in presence of T4 was enhanced significantly in dose related manner.     

Phylogenetic studies on T cells. I. Lymphocytes of the shark with differential response to phytohemagglutinin and concanavalin A

Peripheral blood lymphocytes of the nurse shark (Ginglymostoma cirratum) respond to stimulation by concanavalin A (Con A) as evidenced by increased incorporation of tritiated thymidine. Separation by means of Ficoll-Isopaque yields two or more bands and a sediment, all of which contain lymphocytes responsive to Con A. Only the bottom cells react to phytohemagglutinin (PHA). This reaction cannot be detected in the unseparated lymphocyte population. Thus, only a unique subset of lymphocytes appears to be responsive to PHA and is probably blocked in its response by other cells. The findings suggest that differentiation toward Con A responsiveness may have preceded phylogenetically the responsiveness to PHA. Judging by the requirement for high concentrations of both mitogens the receptor sites on shark lymphocytes appear to be present in lower densities than on lymphocytes of higher vertebrates.     

Separation of human lymphocyte subpopulations by density gradient electrophoresis. I. Different mobilities of T (T mu, T alpha) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.