Characterization of T and B antigen-binding cells for beta-galactosidase. III. Independence of antigen-binding cells in normal animals from antigenic stimulation.

Antigen-binding cells to beta-galactosidase were enumerated in thymus and spleen of mice of different ages and found to remain at a constant frequency throughout life. Thymic beta-galactosidase-binding cells (ZBCs),6 representing T-binding cells, showed no appreciable fluctuation, whereas splenic ZBCs, a mixture of T- and B-binding cells, were slightly depressed at birth but found at normal frequencies by 1 week of age. In addition, germfree mice, both within the 1st week after birth and after maturity, had nearly as many binding cells as did conventionally reared age-matched mice. These results considered together suggest that the ability of cells of both T and B origin to recognize antigen, as revealed by their ability to bind beta-galactosidase, arise independently of specific or nonspecific antigenic stimulation, as part of the normal ontogenic sequence of steps of differentiation. This is consistent with the theory of clonal precommitment of T and B cells.     

The differentiation of T lymphocytes. Density characterisation of thymic and peripheral T cells responding in syngeneic and allogenic mixed lymphocyte reactions.

Equilibrium density separation on continuous albumin gradients was used to separate and characterise the T cells responding by proliferation to both syngeneic and allogeneic stimulating cells in the one-way mixed leucocyte reactions (MLR). In CBA mouse spleen both light and dense T cells were capable of responding in an allogeneic MLR. No T cells responding to stimulation be syngeneic B lymphocytes could be isolated from adult or 7-day CBA mouse spleen. In adult CBA mouse thymus, cells responding to allogeneic stimuli were enriched in the light density region, along with the low theta subpopulation. Self-reactive cells, responding with proliferation when cultured with syngeneic adult CBA splenic lymphocytes, and found in adult and 4-day CBA mouse thymus, were also enriched in the light density zones. However, in adult thymus syngeneic MLR reactivity was also found in the dense zones, and the density distribution profiles of total syngeneic MLR responding cells revealed a series of peaks extending over the whole density range. It was suggested that these syngeneic MLR responders undergo a complete maturation process, including progressive density increases, within the thymus gland. Such a sterile differentiation pathway could be a censorship process, leading to death of self-reactive cells within the thymus.     

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.     

Suppressor T cells in thymus-reconstituted nude mice: regulation of mitogen-induced transformation

A population of glass-wool-adherent splenic cells which could suppress the response of other spleen cell populations to T-cell mitogens was isolated from thymus-reconstituted nude mice. Such adherent cells were characterized as sensitive to anti-Thy 1.2 and complement treatment. Glass-wool-adherent cells from athymic mice do not have suppressor activity to self or normal littermate NAC; however, these mice possess precursor suppressor cells as demonstrated by isolation of glass-wool-adherent T regulatory cells in thymus-grafted nude mice. Such cells are generated in either freshly obtained or in vitro cultured thymus. Evidence for suppressor T cells of host genotype was supported by their sensitivity to host-specific anti-Thy serum treatment as well as their generation in alymphoid thymus grafts. Prior anti-Thy 1.2 treatment of GAC partially removed the suppressor activity: however, macrophages and B lymphocytes were shown not to be secondary regulatory cells or suppressor mediators, thus mature T lymphocytes with low amounts of Thy 1.2 antigen may be responsible for this residual suppression. Further characterization of GAC indicates that active cell growth is required for their regulatory function, as irradiation removed the suppressor activity. This report provides evidence for the presence of a T-lymphocyte subpopulation which has a regulatory function and requires a thymus in the generation of these cells.     

Antibody response to simian virus 40 tumor antigen in nude mice reconstituted with T cells.

Athymic BALB/c nude mice (nu/nu) fail to generate circulating antibodies to simian virus 40 (SV40) tumor (T) antigen when immunized with SV40-transformed mouse cells or with T antigen positive somatic cell hybrids derived from SV40-transformed human and normal mouse parental cells. However, normal BALB/c mice readily produce antibodies to SV40 T antigen. When nude mice were reconstituted with normal syngeneic T lymphocytes from spleen or thymus source, the humoral immune responsiveness to SV40 T antigen was restored.     

Macromolecular insoluble cold globulin (MICG): a novel protein from mouse lymphocytes. IV. Evidence for the plasma membrane distribution of MICG.

Macromolecular insoluble cold globulin is a glycoprotein synthesized predominantly by T lymphocytes in the mouse. The present report details experiments demonstrating the plasma membrane distribution of MICG on T lymphocytes. By utilizing immunofluorescent techniques it was shown that MICG was located in the external cell surface of 98% of thymic lymphocytes and 60% of splenic lymphocytes. Furthermore, in spleen cells, it was demonstrated that T cells and not B cells were surface MICG positive. Antibody to MICG was able to cap all of the immunofluorescent-positive (60%) spleen cells. In contrast, anti-MICG antibody did not induce cap formation on thymus cells. Only when dilute solutions of antibody were used did MICG cap on the thymus cells. Employing limited proteolysis of thymus and spleen cells MICG was shown to be regenerated on the surface of T cells with a half-life of 3.5 hr. The distribution and cell surface characteristics of MICG are discussed in terms of a "receptor-like" function for this protein.     

Avian antigen-binding cells. II. Enrichment of antigen-binding B and T cells.

Antigen-binding B and T cells from chicken spleens were selected on plates of antigen-derived gelatin. Hapten-specific B cells from DNP-immune normal chicken spleens were selected on DNP-gelatin. As much as 165-fold functional enrichment of precursors of anti-DNP antibody-producing cells, as measured in an adoptive transfer system, was achieved. However, the enrichment of DNP-binding cells assessed by rosette formation and autoradiography was no more than 25-fold. HGG-specific T cells from bursectomized agammaglobulinemic chickens immunized with deaggregated HGG were selected on HGG-gelatin. In the fraction adherent to HGG-gelatin, at least a 20-fold enrichment of suppressors of the antibody response to TNP-HGG, as measured by adoptive transfer, was accomplished. In contrast, no more than 6-fold enrichment of HGG-binding cells was detected by autoradiography. Antigen-specific depletion and enrichment of suppressor T cells and of HGG-binding cells occurred in parallel, suggesting that suppressor cells can bind soluble antigen and can be isolated on antigen coupled to a solid support.     

Thymus-repopulating capacity of cells that can be induced to differentiate to T cells in vitro.

It was established previously that committed precursors of T cells, which reside in bone marrow and spleen and lack T cell surface differentiation antigens, can be induced by thymopoietin and certain other agents to differentiate rapidly in vitro into T cells bearing typical surface antigens, including Thy-1 and TL (Komuro-Boyse assay). To relate this differentiative step observed in vitro to physiologic events in vivo, a system was devised to trace the migration of precursor cells to the thymus, and their maturation to T cells. Lethally irradiated mice of a TL- strain received spleen cells from TL+ hybrids i.v., and the TL+ population of the thymus was enumerated 13 to 20 days later. Donor TL+ cells first became detectable at 13 days and increased thereafter. Preliminary tests showed that cells capable of migrating to the thymus have a similar density to the cells that are inducible in the Komuro-Boyse assay, this being lower than that of mature of T cells. The thymus-repopulating properties of the donor spleen population were not affected by: 1) pre-treatment in vitro with thymus extract or thymopoietin, which initiates differentiation of T cells precursors, nor b) pre-treatment with anti Thy-1 serum plus complement, which eliminates differentiated T cells. But pre-treatment a) and b) applied in sequence markedly reduced the capacity of spleen cells to repopulate the thymus. These results can be interpreted as follows: induction of Thy-1-TL- precursor cells (pro-thymocyte) in vitro yields Thy-1+TL+ cells (early thymocytes) which have not yet lost their property of repopulating the thymus; therefore, thymus-repopulation was not depleted by treatment a) alone, which induced Thy-1 +TL+ cells, nor by treatment b) alone, which did not affect thymus-repopulation by Thy-1-TL- cells, although treatments a) plus b) did eliminate the newly induced Thy-1+TL+ cells and thus impaired repopulation of the thymus. We conclude that the cell which responds to thymopoietin in the Komuro-Boyse assay by expressing the T cell surface phenotype is the same cell (pro-thymocyte) that normally migrates in vivo from hemopoietic tissues to the thymus and is there induced by thymopoietin to express the phenotype of an early T cell.     

Requirements for stimulation of T cell responses against virus-infected cells: nature of ectromelia virus-infected cells capable of stimulating cytotoxic T cells in a secondary response in vitro.

The nature of infected stimulator cells in the in vitro secondary cytotoxic T cell response to ectromelia infection was investigated. It was found that macrophages were better stimulator cells than spleen cells. B cells (Ig-positive cells) were superior to T cells (Ig-negative cells) both on a relative proportion and on a cell-to-cell basis. Concanavalin A and lipopolysaccharide-stimulated lymphocytes were also effective stimulator cells but appeared to be slightly inferior to spleen cells. Spleen cells depleted of Ia-positive cells were markedly inferior to normal spleen cells as stimulators. It was also found that primary and secondary cytotoxic T cells were largely Ia-negative. These findings are discussed in relation to the likely events during T cell responses to infection in vivo.     

Characteristics of the immature cells involved in T cell-mediated enhancement of syngeneic tumor growth.

Enhancement of tumor growth was observed when non-sensitized thymocytes were injected together with tumor cells into syngeneic mice, although this tumor enhancement was less pronounced than that caused by tumor-sensitized T lymphocytes. The cells within the thymus which are responsible for this tumor enhancement were found to be rapidly dividing and to be absent from the thymus a day after cortisone administration. At a longer time interval the cortison-depleted thymus was repopulated by dividing cells which exhibited tumor-enhancing reactivity. The characteristics of these cells suggest that they are in the early stages of thymic processing. The enhancing thymocytes were sensitive to treatment with the thymic humoral factor which functions in T cell maturation, and their enhancing activity was cancelled by such treatment. These results are compatible with our hypothesis that exposure of immature T cells to a tumor stimulus may lead to tumor enhancement whereas interaction between mature T lymphocytes and tumor cells may be required for tumor inhibition.     

Purine metabolic enzymes in lymphocytes. I. Adenosine deaminase and purine nucleoside phosphorylase activities in mouse lymphocyte subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA-P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleen of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one-third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA-P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA-P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA-P and Con A, and that of PNP activity was enhanced by PHA-P but not by Con A.     

A human CD2 minigene directs CRE-mediated recombination in T cells in vivo

We have produced a mouse line that expresses the Cre recombinase under the regulation of a human CD2 minigene to facilitate Cre-specific recombination in T lymphocytes. These mice express Cre in thymocytes and T cells and are capable of efficient site-specific recombination as shown in the spleen and thymus, but not other tissues. These mice thus provide an excellent resource for the study of gene function during thymocyte development and in mature T cells.     

Tolerance induced by TNP-derivatized syngeneic erythrocytes: evidence for cooperation between hapten-specific T and hapten-specific B lymphocytes in the immune response.

Tolerance to the DNP haptenic determinant was induced with a single i.v. injection of trinitrophenylated syngeneic red blood cells. The tolerant state lasted 1 month and was stable on transfer to irradiated thymectomized syngeneic recipients. Suppressor activity was found soon after injection of tolerogen but was lost before the termination of tolerance. The unresponsive state could be reversed by adding normal thymus cells to tolerant spleen cells but not by normal bone marrow cells. LPS when given with immunogen restored the normal immune response in tolerant mice. Thus the injection of TNP-MRBC induced partial immune unresponsiveness which was characterized by the induction of T cell suppressor activity and by a hapten-specific helper T cells tolerance. Finally, these studies suggest a cooperative interaction between DNP-specific T lymphocytes and DNP-specific B lymphocytes in the immune response to DNP-BGG.     

Avian antigen-binding cells. I. Characterization of antigen-binding T cells from bursectiomized chickens by autoradiography.

Antigen-binding cells (ABC) from spleens of HGG-immunized, bursectomized agammaglobulinemic (Bx) chickens were detected by direct autoradiography with 125I-HGG and by sandwich autoradiography with HGG plus 125I-goat-anti-HGG. The specificity of antigen binding was demonstrated by 1) inhibition of binding of 125I-HGG by preincubation with unlabeled HGG and 2) a specific increase in ABC after immunization. The ABC from Bx chickens were not B cells, as shown by the virtual absence of immunoglobulin-bearing cells in this population and by the lack of inhibition of antigen binding by anti-immunoglobulin sera. The ABC were not macrophages and did not bind HGG via Fc receptors because their frequency was unchanged after passage over nylon wool or incubation with antigen-antibody complexes. The temperature dependence and azide stabilization of the ABC were characteristic of antigen-binding T cells. Therefore, T cells capable of binding soluble antigen were demonstrated in Bx chicken spleen, which is free of contamination by B cells and passively adsorbed antibody.     

In vitro and in vivo activation of murine gamma/delta T cells induces the expression of IgA, IgM, and IgG Fc receptors.

The present studies examined resting and activated murine gamma/delta T lymphocytes, in vitro and in vivo, for surface expression of FcR. Polyclonal gamma/delta TCR+ lymphocytes selectively grown from the spleen and intestine of normal mice did not express FcR when the cells were in a resting state, but when cells were activated with anti-CD3 antibody virtually all of the splenic gamma/delta lymphocytes and a large subpopulation of the intestinal gamma/delta lymphocytes expressed IgA and IgM FcR. This was confirmed by using transgenic mice. Resting gamma/delta TCR+ lymphocytes from the spleen, thymus, lymph node, and blood of gamma/delta TCR transgenic mice did not express FcR for any of the five major classes of Ig H chains. Activation of the gamma/delta TCR+ cells via the CD3/TCR complex induced high levels of IgM and IgA FcR and low levels of IgG FcR. Finally, in hepatic granulomas of schistosome-infected mice, activated gamma/delta TCR+ cells are present and express high levels of IgA and IgM FcR and low levels of IgG FcR. These investigations establish that transition of gamma/delta TCR+ lymphocytes from a resting to an activated state (triggered via the T3Ti TCR complex) is accompanied by the induction of surface membrane receptors specific for Ig H chain isotypes. The activation-linked expression of FcR on gamma/delta TCR+ lymphocytes provides potential mechanisms for coupling the functional activities of gamma/delta T lymphocytes with immune mechanisms that involve Ig molecules, such as antibody-dependent cellular cytotoxicity.     

Cyclic adenosine 3',5'-monophosphate response to dopamine in mouse lymphoid cells and cell lines

Cyclic adenosine 3',5'-monophosphate responses to dopamine and isoproterenol were studied in mouse and rat spleen, thymus, lymph nodes and Peyer's patches lymphocytes and in 7 mouse cell lines of T- and B-lymphoid derivation. The responses of normal cells to dopamine were moderate, of the same extent, but selective to spleen and thymus in mouse, and to spleen and lymph nodes in rat. The YAC-1 T lymphoma cell line was sensitive to dopamine with a higher magnitude than normal lymphoid cells. Dopamine was less potent than isoproterenol in all cells, and whereas dopamine-sensitive and isoproterenol-sensitive cells, or dopamine-insensitive and isoproterenol-insensitive cells were found, no cell type was dopamine-sensitive and isoproterenol-insensitive. Altogether, these results suggest that only a small subset of lymphocytes is susceptible to the cAMP-elevating action of dopamine.     

Biological characterization of T and B lymphocytes separated from normal mouse spleen by a physical adherence column method

The capacity of spleen cell populations enriched for T and B lymphocytes by a physical adherence column method to respond in vitro to phytomitogens and allogeneic lymphocytes was determined. Column filtrate cells (T lymphocytes) responded well to phytohaemagglutinin- and mitomycin-C-treated allogeneic spleen cells, but poorly to pokeweed mitogen. Adherent cell populations from the column (B and some T lymphocytes) responded well to pokeweed mitogen, but poorly to phytohaemagglutinin- and mitomycin-C-treated allogeneic cells.Purified peripheral T lymphocytes prepared from normal mouse spleen by the column method reconstituted the depleted in vitro antibody response to the thymic-dependent SRBC antigen of all B lymphocyte sources tested, namely, spleen cells from congenitally athymic mice, neonatally thymectomized mice, and adult thymectomized mice which had been reconstituted with bone marrow, and a lymphocyte population prepared by incubating spleen cells with anti-θ serum and complement. When transferred with sheep erythrocytes to congenitally athymic mice, purified peripheral T cells restored the in vivo IgM and IgG responses of these animals. These results confirm that the column filtrate is a thymus derived subpopulation of cells capable of cell-mediated immunity and cooperation with B lymphocytes in humoral immunity both in vitro and in vivo.     

A phenotypic and functional analysis of long-lived B and T lymphocytes

The lymphocyte composition of spleen, lymph nodes, bone marrow, and thymus of mice submitted to hydroxyurea treatments for four consecutive days was studied. The treatment selects for small lymphocyte populations that represent between 4 and 20% of control numbers in the various organs. Spleen and bone marrow contain the same B cell population with a low IgM, high IgD, low I-E phenotype, which respond to LPS at control clonal frequencies. The T cell compartment is equally depleted, and the lymphocytes remaining contain frequencies of clonable cells in response to mitogens and IL-2 that are comparable to those detected in normal spleen cells. Overall, the results suggest that only a minor fraction of all lymphocytes in a normal young adult mouse have life spans longer than 4 days.     

Further studies on Th-B, a cell surface antigenic determinant present on mouse B cells, plasma cells and immature thymocytes.

A goat antiserum (Goat anti-M104E) has been produced which contains antibodies selectively cytotoxic for mouse B cells and a subpopulation of thymus cells. It reacts with the Th-B antigenic determinant which has been shown by us (1–3) to be present on B cells and on plasma cells and on some cells in the thymus. It also is very cytotoxic for mouse B cells while a previously developed rabbit antiserum was not. The antiserum was obtained by immunization with cells of the BALB/c mouse myeloma MOPC-104E. When the antiserum was purified by in vivo absorption in mice, antibodies remained which were cytotoxic for cells of all of several myelomas at a titer between 1:128 and 1:1024 as determined by an in vitro complement dependent cytotoxicity test. The in vivo purified antibodies were also cytotoxic for about 70% of thymus cells, for about 70% of spleen cells, for about 50% of lymph node cells and for about 20% of bone marrow cells. They were very cytotoxic for splenic or lymph node B cells separated from T cells by a nylon wool column and only slightly cytotoxic for splenic or lymph node T cells. The antibodies were only weakly cytotoxic for one out of five T cell tumors tested and not cytotoxic for the remaining four. Irrespective of target cells used, the cytotoxicity of purified Goat anti-M104E was easily removed by absorption with cell suspensions from tissues which contain B cells, plasma cells or thymus cells. In order to confirm that the same anti-Th-B antibodies recognize the determinant present on spleen cells and on some thymocytes, the purified Goat anti-M104E serum was absorbed with either spleen cells or thymus cells. The absorbed sera were tested for ability to label thymocytes or spleen cells using the fluorescence activated cell sorter (FACS). Either absorption removed essentially all the antibody capable of binding to either cell population. In addition it was shown, using the FACS, that only B cells and not T cells of the spleen contain the Th-B determinant. The anti-Th-B antibodies have now been used for the rapid elimination of B cells from a mixed population of lymphocytes without affecting the function of mature T cells. Thus in vitro treatment of spleen cells from SRBC-immunized donors with purified Goat anti-M104E plus complement results in the killing of a high proportion of the B memory cells as shown by the reduction of PFC produced when the treated cells are transferred to irradiated recipients. The T cell helper function of the transferred cells is not affected by Goat anti-M104E treatment as shown by appropriate cell transfer experiments in which effective B cells are provided by an AKR anti-Thy-1.2-treated spleen cell population and effective T cells are provided by the Goat anti-M104E-treated spleen cell population. Antibodies detecting Th-B may serve as an approach to understanding the ontogeny of lymphocytes. Our results suggest that Th-B is a cell surface marker appearing early in the development of lymphoid cells, on the common precursor of B and T cells and that it is lost from T cells as they mature in the thymus.