Immunologic functions of isolated human lymphocyte subpopulations. IV. Stimulation of MLC and CML by human T cells.

Surface immunoglobulin positive and immunoglobulin negative human lymphocyte populations were obtained by immunoabsorbent column chromatography. Both cell populations were effective as stimulating and target cells in allogeneic MLC and CML reactions. The immunoglobulin negative population was further depleted of both EAC rosette forming cells and nylon wool adherent cells. The resulting highly purified T cell population was also able to stimulate allogeneic cells in MLC, and induce the generation of specifically cytotoxic killer cells in CML.     

Immunologic functions of isolated human lymphocyte subpopulations. III. Specific allogeneic lympholysis mediated by human T cells alone.

The studies presented herein have evaluated both the specificity and cellular basis of cell-mediated lympholysis (CML) in man. An efficient and quantitative 51Cr release assay was utilized to study the role of highly purified human T and B cells in CML. After in vitro sensitization human T cells develop the capacity to kill specifically allogeneic cells to which they were sensitized. In contrast, B cells were neither triggered to proliferate nor activated to kill allogeneic targets. B cells were not activated to kill even when sensitized in the presence of potentially "helper" T cells, nor did they block T cells from killing during the effector phase. Cell-free supernatants taken from active in vitro sensitization cultures were not lympholytic and did not modulate T cell killing. Hence, these studies show that both the afferent and efferent phases of human CML are T cell functions.     

Rabbit B spleen lymphocytes and T helper cells. I. Responsiveness to mitogens of B cell subpopulations of different sedimentation velocities and subpopulations bearing or lacking Fcgamma receptors.

The response to anti-allotype (anti-Ab4), Nocardia Water Soluble Mitogen (NWSM), pneumococcal polysaccharide type III (SSS III), and human Fc fragments of various purified and unfractionated rabbit spleen cell populations was determined in terms of 3H-thymidine up-take. B cells were isolated either from untreated suspensions of spleen cells or from suspensions from which adherent and phagocytic cells were removed. The purification factor was greater than the enhancement of 3H-thymidine uptake by anti-Ab4, NWSM, and SSS III as compared with the response of unfractionated spleen cells. It thus appears that a helper cell was involved: the mitogen response of purified B cells was enhanced by the addition of T cells. B subpopulations were separated by sedimentation or by rosetting, which allowed us to separate Fcgamma receptor-bearing cells from cells that did not possess this receptor. There were differences between cells responding to B mitogens not only in sedimentation velocity but also in the absolute number of cells. B cells bearing the Fcgamma receptor were less responsive to anti-Ab4 and more responsive to SSS III, NWSM, and human Fc than were B cells lacking the Fcgamma receptor.     

Lymphoid cell subpopulations. I. Synergy between lymph node cells and thymocytes in response to alloantigens and mitogens.

Mixtures of isogenic thymocytes (TC) and lymph node cells (LNC) were shown to exhibit synergistic responsiveness to M and H-2 alloantigens in the mixed lymphocyte interaction (MLI). With respect to the kinetics and magnitude of proliferation and effector cell generation, the response occurring in synergizing cultures closely resembled that of optimal numbers of LNC or spleen cells (SC). In addition, the antigen specificity of effector cells generated by synergizing cultures was similar to that of effectors derived from cultures containing optimal numbers of responding SC. LNC-TC mixtures also exhibited synergy in response to the phytomitogens concanavalin A and pokeweed mitogen but not to phytohemagglutinin. Weakly positive synergy was observed in response to bacterial lipopolysaccharide. It is proposed that the phenomenon of synergy is not restricted to cultures containing mixtures of LNC and TC but also occurs in cultures containing optimal numbers of LNC or SC as a result of interactions between subpopulations of lymphocytes contained within these tissues.     

Separation of human lymphocyte subpopulations. I. Transformation characteristics of rosette-forming cells

Lymphocytes were isolated from human peripheral blood by carbonyl-iron treatment and Ficoll-Isopaque centrifugation. The lymphocytes were allowed to form rosettes with sheep erythrocytes, either uncoated (E) or coated with antibody and complement (EAC).In 32 experiments E rosettes were separated from free lymphocytes on a Ficoll density gradient. Thus, depleted (non-E) and enriched (E) fractions were obtained. It was found that in the original suspension 24 ± 7.2% of the lymphocytes formed rosettes with EAC and 56 ± 8% with E. In fraction non-E these values were 56 ± 11.4 and 22 ± 8.9%, respectively; in fraction E 8 ± 3.8 and 79 ± 8.8%. Moreover, the percentages of Ig-bearing cells among the fractions were found to follow closely those of CRL.In a series of lymphocyte culture experiments these fractions were compared with the original suspension and a control suspension (rosetted, not separated), as well as with a recombined population (non-E + E). It was found that fraction non-E showed an increased response to PHA and PWM, and an enhanced MLC stimulatory capacity, whereas fraction E was decreased in these respects. Moreover, fraction E displayed significantly reduced spontaneous DNA synthesis.It is concluded that the responses to PHA or PWM, as well as the capacity to stimulate allogeneic cells, are not solely dependent on the cells forming rosettes with E.     

Quantitative analysis of neutral glycosphingolipids from human lymphocyte subpopulations.

The glycosphingolipids of normal human lymphocytes from individual donors were analysed by high-pressure liquid chromatography. In addition, purified T- and B-lymphocytes were examined separately. Lactosylceramide was shown to be the major neutral glycosphingolipid in human lymphocytes, and monohexosylceramide, trihexosylceramide, globoside and paragloboside were all detected in smaller amounts. Analysis of purified B- and T-cell fractions revealed that each of these populations contained a similar qualitative profile for neutral glycosphingolipids, but that quantitatively, B-cells contained several times more of each glycosphingolipid per cell than did T-cells.     

Rabbit lymphocyte subpopulations. I. Separation of Ig+ and Ig- cells and their interaction in cultures stimulated by mitogens.

Rabbit lymph node cells (Ig⁺Ig^?) were rosetted with anti-Ig antibody-coated erythrocytes and the rosetted Ig⁺ cells (B cells) were separated from unrosetted Ig^? cells (T cells) by centrifugation through Ficoll-Hypaque medium. The Ig^? cells were recovered from the top and the Ig⁺ cells from the bottom of the Ficoll-Hypaque layer. Some of the purified Ig⁺ cells lost their ability to form rosettes when cultured with the mitogen associated with streptolysin O. This suggested that the Ig⁺ population might contain two distinct subpopulations. The response of Ig⁺Ig^?, Ig⁺, and Ig^? cells to various mitogens was studied. The Ig^? cells incorporated more ³H-TdR when they were incubated by themselves than when they were cultured with Ig⁺ cells in an Ig⁺Ig^? culture. On the other hand, the Ig⁺ cells incorporated less ³H-TdR when they were incubated by themselves than when they were incubated with Ig^? cells in an Ig⁺Ig^? culture. Thus, Ig⁺ cells suppressed the response of Ig^? cells whereas Ig^? cells enhanced the response of Ig⁺ cells. We conclude that rabbit Ig⁺ cells (B cells) and Ig^? cells (T cells) interact with a feedback pattern of regulation.     

Functional properties of lymphocyte subpopulations in hepatitis B virus infection. I. Suppressor cell control of T lymphocyte responsiveness

Hepatocellular injury in hepatitis B virus infection may be produced by an autoaggressive hepatocytotoxic immune response. To test the hypothesis that acquired suppressor cell defects may participate in such a response, we assessed the functional integrity of 2 suppressor cell populations in patients with type B viral hepatitis. Spontaneous suppression of the 1-way mixed lymphocyte response by radiation-resistant, adherent peripheral blood mononuclear cells decreases during the acute phase of disease, returns towards normal with clinical recovery, but remains depressed in patients with chronic hepatitis. The degree of spontaneous suppressor cell dysfunction correlates inversely with at least 1 biochemical parameter of hepatocellular injury (SGPT). The functional integrity of this suppressor cell fluctuates during chronic hepatitis and may reflect currently undefined biologic variables in this disease. Mitogen-induced suppression on lymphocyte activation by radiation resistant, nonadherent suppressor cells is also depressed in acute and chronic hepatitis, but it does not correlate with biochemical evidence of hepatocellular injury on an individual-patient basis. Documentation of these generalized defects of nonspecific suppressor cell function establishes a basis for the possible existence of specific anomalies of immuno-regulation that may permit the expression of normally suppressed auoaggressive hepatocytotoxic immune mechanisms in viral hepatitis.     

The cytolytic T lymphocyte response to trinitrophenyl-modified syngeneic cells. I. Evidence for antigen-specific helper T cells.

Mice were primed subcutaneously with trinitrophenyl (TNP)-modified syngeneic spleen cells. Seven days later, spleen cells from these in vivo primed mice, or spleen cells from naive mice, were co-cultured with TNP-modified syngeneic cells. Spleen cells from the in vivo primed mice demonstrated augmented cytolytic T lymphocyte (CTL) activity. The spleens of these in vivo primed mice contained a population of radioresistant, antigen-specific, helper T cells. Specifically, spleen cells from these mice, after x-irradiation, were able to augment the in vitro CTL response of normal spleen cells to TNP-modified syngeneic cells.     

Histamine suppression of human lymphocyte responses to mitogens.

Exogenously added histamine in non-cytotoxic concentrations (10^?5?10^?3M) suppresses in vitro proliferation of lymphocytes induced by PHA or Concanavalin A. This suppressive effect was observed when histamine was present for as short as $\text{1}\text{2}$ hr in the beginning of the culture. Histamine, in concentrations as high as 10^?3M, did not cause increased release of isotope from ⁵¹Cr-labeled lymphocytes following 4 hr of incubation. The histamine H₂ receptor antagonist, metiamide, but not the H₁ receptor antagonists diphenhydramine or chlorpheniramine, blocked the histamine suppressive effect. Some of the biological implications of these findings are discussed.     

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.     

Potentiation of the T lymphocyte response to mitogens IV. Serum-free production and testing of macrophage soluble products.

Serum-free cultures of activated macrophages generate conditioned media containing both potentiating and inhibitory activities for the lectin-induced transformation of syngeneic thymocytes, lymph node cells, or spleen cells, also cultured in serum-free medium. Exhaustive dialysis of macrophage conditioned medium (MCM) eliminates its inhibitory activity. At mitogenic doses of lectin, the dialyzable material enhances the potentiating activity exerted by macro-molecular factors at low and optimal concentration of MCM. The inhibitory effect of intermediate concentrations of nondialyzed MCM on [³H]thymidine uptake can be reversed if the cells are washed and pulsed in fresh medium, and thus is artefactual. On the other hand, high doses have a real inhibitory effect on proliferative response of transformed lymphocytes. Rat MCM is not mitogenic for any of the target lymphocytes tested. Its effect is observed both in primary cultures of lymphocytes and secondary cultures of blast cells.     

Polyclonal activation of human lymphocytes in vitro. I. Characterization of the lymphocyte response to a T cell-independent B cell mitogen

The staphylococcal cell wall component protein A (SpA) and formalinized, Cowan I strain Staphylococcal organisms (STA) were compared with the lectins phytohemagglutinin, concanavalin A, and pokeweed mitogen for their ability to trigger proliferation of normal human lymphocytes, lymphocyte subpopulations, and cells from patients with primary immune deficiency diseases. SpA was found to be a potent T cell mitogen, very similar to the other lectins tested. It failed to stimulate purified non-T cells and peripheral blood lymphocytes from patients with different forms of severe combined immunodeficiency disease (SCID). STA, treated to prevent the leakage of soluble SpA during culture, exclusively stimulated non-T cells: the responding cell population was characterized to be E-rosette negative but positive for C3 receptors, surface Ia, a receptor for STA itself, and likely carried surface immunoglobulin. Normal responses to STA were found in patients with the adenosine deaminase-positive form of SCID. In 18 patients with humoral immune deficiency syndromes, the presence of STA responses was correlated with the presence of circulating, surface immunoglobulin-bearing cells. A commercial STA preparation was rendered B cell specific after reformalinization, a procedure that eliminated the shedding of soluble SpA under culture conditions.     

Peanut agglutinin. I. A new tool for studying T lymphocyte subpopulations.

Fluorescein-coupled peanut agglutinin (PNA) has been used at the single-cell level to study mouse lymphocyte subpopulations. PNA not only binds to most thymocytes, as has already been shown by other authors, but also binds to a small fraction of peripheral lymphocytes that are all T cells (theta+Ig-) or null cells (theta-Ig-). Most PNA-positive thymocytes are sensitive to in vivo corticosteroids and irradiation (450 rads) treatments. Conversely, the positive spleen cells (5% of total spleen lymphocytes) are essentially resistant to corticosteroids and irradiation. Study of PNA binding during ontogenesis shows the occurrence of PNA-positive cells in the fetal liver before thymus constitution and in the very beginning of embryonic thymus and spleen development. These data indicate that PNA is a marker of early T cell subpopulations but that there are probably several distinct subsets of PNA-positive T cells.     

Production of a human T lymphocyte chemotactic factor by T cell subpopulations

Concanavalin A-stimulated human peripheral blood mononuclear cells release a lymphocyte chemotactic factor. This lymphocyte chemotactic factor is produced optimally after 24 to 48 hr of culture and is not found before 3 hr of culture, which suggests that the factor is synthesized de novo and is not preformed and secreted after Con A stimulation. This is further supported by experiments showing that the protein synthesis inhibitors cycloheximide and puromycin totally prevent the production of the chemotactic factor. Experiments using cultured and uncultured T lymphocytes as responding cells show that cultured T cells respond more efficiently than uncultured T cells to this factor. Furthermore, the lymphocyte chemotactic factor preferentially stimulates T lymphocyte locomotion as compared to peripheral blood non-T lymphocyte migration. Fractionation of mononuclear cells into glass nonadherent lymphocytes, monocyte-enriched preparations, T lymphocytes, and non-T lymphocytes shows that lymphocyte chemotactic factor is produced by Con A-stimulated, glass nonadherent lymphocytes and T cells but not by monocytes or non-T lymphocytes. Further fractionation of T lymphocytes into Leu-2 and Leu-3 T cell subpopulations shows that the production of T lymphocyte chemotactic factor can be attributed to the Leu-2 suppressor/cytotoxic T cell subset. The generation of a T lymphocyte chemotactic factor by Leu-2 T cells may represent a means of recruiting other T cells to the site of its release.