Homologous antigen for T cell receptor in axial organ cells from the asterid Asterias rubens.

The axial organ (A.O. cells) of the sea star Asterias rubens is a primitive immune organ. The total population was fractionated into two populations: adherent (B-like) and non-adherent (T-like) to nylon wool. The adherent cells resemble mammalian B lymphocytes and bear homologous human T cell receptor (beta chain) to a higher degree than T-like cells which resemble T lymphocytes.     

Immunology: the origin of sweetbreads in lampreys?

The thymus is required for the differentiation of T lymphocytes. A new study in lampreys indicates that the pharyngeal epithelium of the gill basket supports the development of T-like cells, suggesting the existence of a primitive thymus in these oldest of vertebrates.     

T-cell development in the absence of a thymus: the number, the phenotype, and the functional capacity of T lymphocytes in nude mice

A small but definite proportion of T-lymphocyte-like cells have been reported in nu/nu (nude) mouse spleen despite the congenital absence of a thymus in these animals. We have determined the number and the characteristics of such cells using flow cytometry. The level of T-like cells increased with age. In 4-month-old nu/nu CBA spleen, 14% of all cells expressed some Thy 1 antigen. However, only 4% expressed mature T-cell levels, and only the 2% with the highest Thy 1 also showed a normal distribution of Ly 1 and Ly 2 antigens. These T-like cells were slightly larger than normal nondividing T lymphocytes. We have assessed the total functional capacity of T-like cells in nu/nu CBA spleen using a high-cloning-efficiency limit-dilution culture system. Almost all precursor cells capable of forming clones when stimulated with concanavalin A in the presence of irradiated spleen cells and growth factors, and almost all precursors of those clones that were cytolytic in a lectin-mediated tumor-cell-lysis assay, were within this 2% subpopulation of nu/nu spleen cells with mature T-cell markers. Increased levels of purified interleukin 2 failed to induce further precursor function, indicating that maturation of pre-T cells was not obtained. However the nu/nu spleen cells bearing mature T-cell markers displayed only 10-30% of the cloning efficiency of normal splenic T cells. The majority of nu/nu spleen T-like cells, even within this phenotypically "normal" subset, appeared to be nonfunctional. We conclude that the absence of a thymus leads to qualitative, as well as quantitative, deficiencies in the T-cell population, and various interpretations are discussed.     

In vitro effect of rabbit anti sea star lymphocyte serum on axial organ cells

The axial organ (AO-cells) of the sea star Asterias rubens is a primitive immune organ. The total population was fractionated or not into two populations: adherent (B-like) and non adherent (T-like) to nylon wool. Rabbit anti sea star lymphocyte serum induces the proliferation of axial organ cells. The T-like antiserum stimulates the T-like cells exclusively; the whole axial organ cell antiserum only stimulates the whole axial organ cell population.     

Immunological importance of the second gut segment of carp.

Lymphocytes, plasma cells, granulocytes (three to four types), macrophages and monocyte-like cells were ultrastructurally distinguished in the intestinal mucosa of carp. Neutrophilic granulocytes and lymphoid cells were present in and under the epithelium throughout the gut. In contrast to macrophages which dominated in the epithelium of the second segment, basophilic and eosinophilic granulocytes (and their intermediates) were mainly found in the connective tissue of the first segment. Applying monoclonal antibodies against serum immunoglobulin (Ig) in an immunogold technique, only a minority of lymphoid cells appeared to be Ig-immunoreactive at their external membrane, suggesting the presence of many more T than B cells in the intestinal mucosa. Except for cells which resembled immature plasma cells, plasma cells did not show, or hardly showed, Ig at their surface. In contrast with the head kidney, plasma cells with an Ig-immunoreactive cytoplasm were scarce in the intestinal mucosa. As mucosa plasma cells were regularly found with the electron microscope, they possibly contain another class of Ig. Macrophages and monocyte-like cells were also found to be Ig-immunoreactive, suggesting the presence of immune complexes at their external membrane. The immunological significance of B- and T-like lymphocytes next to immune complex-binding and antigen-presenting macrophages in the second gut segment is discussed.     

The thymus in fish: Development and possible function in the immune response

With the exception of Agnatha, fish possess the functional equivalent of the thymus gland found in higher vertebrates. As in other vertebrates, this gland originates from the pharyngeal pouches and ontogenically is the first lymphoid organ to be infiltrated with lymphoid cells. Histology of the structure may differ from one species to another but the cellular component is basically similar. The (paired) gland is surrounded by an epithelial capsule. Within the gland a framework of reticulo-epithelial cells supports the lymphocytes. The age-related involution process, which characterizes the thymus of higher vertebrates, does not necessarily occur in fish. Nevertheless, thymus growth and function may be modulated by those factors that induce its involution such as aging, season, sexual maturity, and stress. The major role played by the thymus in the immune response of higher vertebrates is presumed to occur in fish. Thymus-derived cell dependent immune reactions have been demonstrated in fish. The cells that mediate these functions are designated as T-like cells. So far, cell surface markers equivalent to those of mammalian T lymphocytes have not been characterized. The T lymphocyte specificities are supposed to be acquired within or via the thymic microenvironment. Unfortunately, there is limited data concerned with the cytological and physiological basis of the maturation of thymus-derived cells. Direct involvement of the fish thymus in defense mechanisms has not been investigated extensively. The gland appears to be weakly protected because of its superficial location and is easily exposed to pathogens. Neoplasia is the main pathologic condition reported in the thymus of fish, with little else having been published regarding thymic pathology.     

Immune Related Genes Underpin the Evolution of Adaptive Immunity in Jawless Vertebrates

The study of immune related genes in lampreys and hagfish provides a unique perspective on the evolutionary genetic underpinnings of adaptive immunity and the evolution of vertebrate genomes. Separated from their jawed cousins at the stem of the vertebrate lineage, these jawless vertebrates have many of the gene families and gene regulatory networks associated with the defining morphological and physiological features of vertebrates. These include genes vital for innate immunity, inflammation, wound healing, protein degradation, and the development, signaling and trafficking of lymphocytes. Jawless vertebrates recognize antigen by using leucine-rich repeat (LRR) based variable lymphocyte receptors (VLRs), which are very different from the immunoglobulin (Ig) based T cell receptor (TCR) and B cell receptor (BCR) used for antigen recognition by jawed vertebrates. The somatically constructed VLR genes are expressed in monoallelic fashion by T-like and B-like lymphocytes. Jawless and jawed vertebrates thus share many of the genes that provide the molecular infrastructure and physiological context for adaptive immune responses, yet use entirely different genes and mechanisms of combinatorial assembly to generate diverse repertoires of antigen recognition receptors.     

Paralysis of street rabies virus-infected mice is dependent on T lymphocytes.

Street rabies virus (SRV)-infected T-lymphocyte-deficient (nude) mice, in contrast to euthymic mice, did not develop hindlimb paralysis prior to death. To document the role of T lymphocytes in rabies virus-associated paralysis, 10(8) spleen cells from normal immunocompetent euthymic mice were transferred to nude mice and the recipient mice were challenged with SRV. One hundred percent of the reconstituted mice developed paralysis and died. Depletion of T cells from the donor spleen suspension prior to transfer abrogated the development of paralysis but did not prevent the deaths of the recipient animals. Mice receiving 10(8) rabies virus-immune spleen cells did not become paralyzed and did not die. Nude mice inoculated with either rabies virus-immune or normal mouse serum prior to and following SRV inoculation did not develop paralysis. Immune serum protected the mice, whereas animals inoculated with normal serum died. Central nervous system inflammatory responses in nude mice immunologically reconstituted with normal spleen cells were characterized by diffuse cellular infiltrates in the parenchyma and extensive perivascular cuffing. Perivascular infiltrates included CD8+ and CD4+ T lymphocytes and Mac-1+ macrophage-microglial cells. Inflammatory cells in the parenchyma were limited to CD8+ lymphocytes and Mac-1+ cells. These observations indicate that paralysis of SRV-infected mice is dependent on T lymphocytes. Whether injury leading to paralysis is mediated by T lymphocytes or by an influence of T lymphocytes on macrophage-microglial cells or other cells remains to be determined.     

Characterization of an antiserum to guinea pig antithrombin III (AT III). II. Stimulation of T lymphocyte proliferation

A goat antibody specific for an antigenic determinant shared between guinea pig antithrombin III (AT III) and thymocytes was shown to be mitogenic for lymph node T lymphocytes in the presence of macrophages. Although the antiserum was not mitogenic for purified populations of B lymphocytes, B lymphocytes were as efficient as T lymphocytes in absorbing the mitogenic activity of the serum. The shared antigenic determinant appeared to be carbohydrate in nature in that native and guanidine-treated AT III, but not periodate oxidized AT III, were capable of inhibiting the mitogenic activity of the serum when added continuously to the cultures. The possibility that the plasma protease inhibitor AT III or an antigenically related membrane protein are involved in the regulation of T cell activation is discussed.     

In vitro effect of silica on axial organ cells in the presence and absence of mitogens

The axial organ (AO-cells) of the sea star Asterias rubens is a primitive immune organ. The total population was fractionated into two populations: adherent (B-like) and non-adherent (T-like) to nylon wool. Mitogenic responses were tested in the presence or absence of silica. Silica inhibits specifically the action of phagocytes contained in T-like and B-like subpopulations. Mitogenic responses do not occur in the presence of silica.     

Nude mice injected with DNA released by antigen stimulated human tlymphocytes produce specific antibodies expressing human characteristics

Nude mice were injected with DNA purified from the nucleoprotein complex released by T lymphocytes previously exposed in vitro to inactivated herpes or poliovirus. After five days the serum of these mice was tested for its virus neutralizing activity. Results show that injected nude mice synthesize antiherpetic or antipolio antibodies depending on the antigen used to sensitize the T lymphocytes in vitro. These antibodies were not found in the serum of uninjected control mice or mice injected with inactivated herpes or polio viruses. Mice injected with DNA release by human T cells produced antibodies carrying human allotypes since they could be neutralized by anti-allotype sera. Moreover their antiviral activity was inhibited by anti-human IgM or IgG. However, the mice which were injected with DNA released by antigen stimulated murine T lymphocytes produced antiviral antibodies which were not neutralized by anti-human allotype sera.     

Locomotion of human lymphoid cells. I. Effect of culture and con A on T and non-T lymphocytes.

Human peripheral lymphocytes were separated from whole blood on a Ficoll-Hypaque gradient. They were then depleted of monocytes, separated into T and non-T fractions, and assayed for locomotor responses toward casein and endotoxin-activated serum in Boyden chambers. Non-T cells showed higher random motility than did T cells. Culture prior to assay was necessary in order to demonstrate locomotor activity of T cells, but this requirement, although desirable, was not essential for non-T lymphocytes. It was not necessary for Con A to be present in the culture medium or for either T or non-T lymphocytes to be in blast form to show locomotion.     

Alpha-fetoprotein and human lymphocyte subpopulations.

Peripheral blood lymphocytes were incubated with varying concentrations of alpha-fetoprotein and enumerated for total and "active' T lymphocytes, B lymphocytes, and their proliferative responses to phytohemagglutinin. No significant effect was observed on total T or B lymphocyte proportions. However, there was a dose-related increase in proportions of the so called "active" T lymphocytes. The response of human lymphocytes to phytohemagglutinin was markedly depressed. The alteration in the proportion of active T cells and the inhibition of T lymphocyte response to phyto hemagglutinin by alpha-fetoprotein occurred at higher concentrations than are present in amniotic fluid, serum of pregnant women, or serum of adults, but well within the range reached in fetal serum. The immunoregulatory role of alpha-fetoprotein is discussed.     

Comparison of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes

Precursors and effectors of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes are compared. Natural killer cells are resistant to gamma-irradiation (1000 R) whereas precursors of lymphokine-activated killer cells and cytotoxic T lymphocytes are sensitive. Lower doses of gamma-irradiation (500 R) remove precursors for cytotoxic T lymphocytes but not lymphokine-activated killer cells. In addition, lymphokine-activated killer cells are regenerated before classical CTL after sublethal doses of gamma-irradiation. Natural killer cells are resistant to anti-Thy 1 and C' and anti-thymocyte serum, but sensitive to anti-asialo GM1 and complement. Precursors of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Precursors of lymphokine-activated killer cells are partially sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Effector cells of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and resistant to anti-asialo GM1 and complement. Lymphokine-activated killer cell effectors are sensitive to anti-asialo GM1 and complement at 24 hr after activation. These effectors are more closely aligned with classical natural killer effectors. Lymphokine-activated killer effectors, 7 days after activation, are resistant to anti-asialo GM1 and complement and sensitive to anti-Thy 1 and complement. Relationships and differences among these cytotoxic subsets are discussed.     

Separation of a population of human T lymphocytes that bind prostaglandin E2 and exert a suppressor activity

Prostaglandin E2 (PGE2) is a potent inhibitor of immune functions. Two possible mechanisms of PGE2-mediated suppression have been proposed: one is a direct inhibitory effect exerted on interleukin 2-producing T cells; the second is mediated by the activation of nonspecific suppressor T lymphocytes. We previously showed that PGE2 can directly activate human T lymphocytes to suppress lymphocyte proliferation and B lymphocyte maturation. Herein is described the binding of 10 to 30% of human peripheral blood T lymphocytes to insolubilized PGE2 coated to albumin-Sepharose. The T lymphocytes that bound PGE2 (PGE2(+] could be eluted by the addition of serum and gentle shaking of the beads. The following data indicated the specificity of the binding: i) T lymphocytes after an overnight incubation, a condition known to abolish sensitivity to PGE2, lost their affinity for PGE2; ii) preincubation of T lymphocytes with PGE2 blocked the binding; iii) PGE2(+) T cells bound PGE after a 24-hr incubation, whereas PGE2(-) T cells did not. Few T cells bound albumin, and only a small percentage (7 to 9%) bound 6-keto-prostaglandin F1 alpha-coated beads. Among PGE2(+) T cells, there was a slight increase in the percentage of OKT8+ cells. Although T cells that had no affinity for PGE2 (PGE2(-] proliferated as well as unseparated T lymphocytes when stimulated with mitogens or antigens, the proliferative response of the PGE2(+) subset was poor. Moreover, PGE2(+) T lymphocytes did exert a strong suppressor activity on mitogen- or allogeneic cell-induced lymphocyte proliferation as well as on pokeweed mitogen-driven B cell maturation into Ig-containing cells. PGE2(-) T lymphocytes were shown not to exert a significant suppressor activity in these assays. The PGE2(+) subset-mediated suppression was not secondary to a carry-over of PGE2 released from the beads, because its suppressor activity was not altered by the addition of an anti-PGE2 serum. Moreover, PGE2(-) T lymphocytes were not sensitive to the inhibitory activity on cell proliferation of PGE2. These results indicate that a given functional subset of peripheral blood T lymphocytes binds PGE2, and that at least some of them are activated into suppressor T cells. The relationship between the PGE2-activatable T suppressor subset and other functionally defined suppressor T cells remains to be clarified; it is suggested, however, that PGE2 can act as an immunoregulator through the activation of identifiable suppressor T cells.     

Identification of membrane-associated lymphotoxin (LT) on mitogen-activated human lymphocytes using heterologous anti-LT antisera in vitro.

Surface-associated lymphotoxin (LT) molecules have been identified on mitogen-activated human lymphocytes employing heterologous anti-α-LT serum in vitro. These membrane-associated LT molecules are present on PHA- or Con A-activated lymphocytes but do not appear to be expressed on unstimulated cells. Furthermore, these molecules were detected primarily on activated T lymphocytes, with little detectable on activated B- or null-cell populations. The removal of surface LT-bearing lymphocytes, using anti-α-LT serum + C′, does not dramatically affect the capacity of the remaining cells to release LT after mitogen restimulation. In addition, the presence of toxic molecules on the surface of activated lymphocytes suggests that these materials may be expressed in an inactive, noncytotoxic form.     

Synthesis of immunoglobulin by rabbit lymphocytes in vitro in response to anti-immunoglobulin antiserum

Stimulation of synthesis of immunoglobulin (Ig) in vitro by Con A and anti-Ig in cultures of rabbit lymphoid cells has been analyzed qualitatively using an assay that measures the incorporation of [³H]leucine into newly synthesized proteins, followed by the specific absorption of tritiated immunoglobulin by staphylococcal protein A. Whereas Con A stimulates Ig production by spleen cells only if T lymphocytes are present, anti-immunoglobulin serum enhances Ig synthesis in the absence of T lymphocytes. In contrast, neither Con A nor anti-immunoglobulin serum stimulates peripheral blood lymphocytes to produce enhanced levels of Ig. It is concluded that both Con A and anti-immunoglobulin serum do not activate resting B cells but drive differentiation of B cells which are already synthesizing Ig. Anti-Ig acts directly whereas stimulation of B-cell Ig synthesis by Con A occurs indirectly through stimulation of T cells.     

In vivo and in vitro regulation of IgE production in murine hybridomas

Normal BALB/c mice injected i.p. with the IgE-secreting hybridomas B53 (epsilon, kappa anti-DNP), SE1.3 (epsilon, kappa, anti-arsonate) or A3B1 (epsilon, kappa, anti-TNP) were monitored for serum IgE concentrations and frequencies of splenic T lymphocytes with surface membrane receptors for the Fc portion of IgE (Fc epsilon R+ T lymphocytes). Mice with B53 or SE1.3 hybridomas initially developed high concentrations of IgE and CD8+ Fc epsilon R+ T lymphocytes, followed by a progressive decline in both serum IgE and expression of cytoplasmic epsilon-chains in the hybridoma cells. Serum IgE concentrations in mice with A3B1 hybridomas progressively increased without development of Fc epsilon R+ T lymphocytes nor a subsequent decline in IgE or change in cytoplasmic epsilon-chain expression in the A3B1 cells. An in vitro system in which the IgE-secreting hybridoma cells were cocultured with spleen cells harvested from mice with established B53 tumors was used to investigate the mechanisms involved in the inhibition of IgE production by the hybridoma cells. The results of these studies indicate that: 1) the induction/upregulation of Fc epsilon R on CD8+ T lymphocytes in vivo requires factors in addition to high serum IgE concentrations; 2) in addition to CD8+ Fc epsilon R+ T lymphocytes and monocytes, another, as yet unidentified, splenic cell component appears to contribute to the process by which epsilon-chain expression in IgE-secreting hybridoma cells is suppressed, and 3) a hybridoma (A3B1) that fails to induce CD8+, Fc epsilon R+ T lymphocytes in vivo and is not inhibited in IgE expression in vivo, nonetheless is inhibited in IgE expression in vitro when cocultured with spleen cells from mice with B53 tumors.     

Distinctive functional properties of human blood L lymphocytes: a comparison with T lymphocytes, B lymphocytes, and monocytes.

Human blood lymphocytes with high affinity Fc receptors have been operationally named L lymphocytes because of membrane-labile IgG markers. L lymphocytes lack membrane-incorporated immunoglobulin and do not form rosettes with sheep red blood cells coated with IgM antibody and mouse complement. These lymphocytes are capable of binding IgG in normal human serum at 4 degrees C and will form rosettes with human lymphocytes coated with Ripley IgG. In this study, functional in vitro properties of isolated L lymphocytes were compared with T lymphocytes, B lymphocytes, and monocytes. To obtain these mononuclear populations, first, plastic adherent monocytes were harvested. T lymphocytes were then isolated by centrifugation of E rosette-forming cells, and other rosetting techniques were employed to isolate L and B lymphocytes by negative selection. The functional properties of L lumphocytes were completely unlike those of T cells, B cells, or monocytes. L lymphocytes did not proliferate in response to mitogens, soluble antigens, or cell surface antigens. Moreover, this population could not replace monocytes in helping T lymphocytes respond to concanavalin A and pokeweed mitogen. Once T cells were supplemented with monocytes, however, the addition of L lymphocytes to the culture greatly enhanced the T lymphocytes proliferative response to phytohemagglutinin, concanavalinA, purified protein derivative (PPD), and streptokinase/streptodornase. L lymphocytes were not a subset of B cells. They did not spontaneously develop surface Ig in culture, and pokeweek mitogen could not induce them to transform and generate cytoplasmic Ig detectable by immunofluorescence. Mixtures of B cells and T cells responded to pokeweed mitogen better than do T cells alone. In contrast, enhanced reactivity with L and T cell combinations was not observed. Another sharp difference between these two populations was the stimulator capacity of each in mixed lymphocyte culture. When B and L lymphocytes were carefully monocyte-depleted, only B cells were effective stimulators of autologous and allogeneic lymphocytes. In comparison with T cells, B cells, and monocytes, L lymphocytes were the only effective killers of human blood lymphocytes sensitized with IgG. L lymphocytes, then, have cytotoxic potential, but cannot proliferate in response to various stimulants or become antibody-producing cells. These findings suggest that L lymphocytes comprise a third lymphocyte population.     

The role of insulin in the response of murine T lymphocytes to mitogenic stimulation in vitro

The ability of insulin to influence the responsiveness of murine T lymphocytes in a culture system containing a serum substitute was documented. The presence of insulin was found to enhance the concanavalin A (Con A) reactivity of the lymphocytes. Once the cells were activated by a short-term exposure to Con A, insulin was capable of replacing Con A for the continued stimulation of the cells. This was true both for lymphocyte proliferation and for the generation of nonspecific cytotoxic T lymphocytes. The presence or absence of insulin was not found to influence the phytohemagglutinin responsiveness of the T lymphocytes. Possible reasons for the observed results are discussed in relation to a proposed model for lymphocyte activation.