Interactions and quantitative analysis of immunoregulatory cells in the chicken thymus

Step-wise dilution of chicken thymus cell suspensions has been used to sequentially reveal suppressor, effector, and helper cells in these suspensions. The cells were tested either alone or in autologous mixture combinations with peripheral blood lymphocytes (PBL) as a source of effector cells. The assays studied were graft-vs-host reaction (GvHR) and mixed lymphocyte (MLR) reaction, spontaneous cellular cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and mitogen responsiveness to Con A, PHA, and PWM. When tested alone, high numbers of thymus cells (1 X 10(7) gave weak or low responses, with the exception of GvHR, which was high. When this number of thymocytes was mixed with a strongly responding PBL effector population, there was marked suppression of the latter. Nonspecific crowding was excluded as a cause for the decreased responsiveness, and the data therefore demonstrated the presence of suppressor cells in the thymus. With gradual reduction of the thymus cell number in the mixtures, the suppressor activity was lost, but concomitant with this was the appearance of, or a gradual increase in, thymus effector cells giving good responses. Further dilutions of the thymus (to, e.g., 1 X 10(5) cells) depleted the suspension of effector cells, but helper cells capable of markedly amplifying the effector potential of PBL were revealed. The suppressor/helper function of the thymus was not only dependent on the absolute numbers of thymus cells present, but also on the degree of inherent responsiveness of the effector PBL. If the response of PBL alone was strong, a thymus suspension containing both helper and suppressor cells (e.g., 1 X 10(6) cells) caused suppression of the PBL; if the PBL alone were weak, this same thymus cell suspension caused enhancement. The outcome of an immune response is therefore dependent not only on the presence or absence of particular cell types, but also on the ratios between these cells. An imbalance in these ratios in vivo may underlie diseases of immunologic origin, e.g., autoimmunity.     

Strain-dependence and cellular aspects of the acceleration of age-dependent shift in class-specific helper and suppressor activity in the thymus of MRL/Mp mice by the LPR gene

Previous studies of the immunoregulatory activity of thymocytes from SJL/J mice have shown loss of suppressor activity for the antibody response by 24 weeks of age with appearance of helper activity. At the same time, suppressor cells developed which inhibit the generation of cytotoxic T lymphocytes (CTL). We now show a similar pattern of helper and suppressor activity in MRL/Mp mice. Presence of the lpr/lpr genotype significantly accelerated the onset of these changes in thymocyte activity. A similar pattern of thymocyte activity was not detected in C57B1/6 mice. In aged MRL-lpr mice, evidence of increased suppressor cell activity for the CTL response could be demonstrated in spleen, and the suppressor was sensitive to treatment with anti-thy 1.2 + complement. The magnitude of the deficiency in the CTL response in MRL-lpr mice was greater than could be accounted for by suppressor cell activity alone. Measurement of the frequency of CTL precursors (CTLP), the yield of CTL per CTLP, and the ability to produce and to respond to interleukin 2 (IL-2) indicated that a drop in CTLP frequency, subnormal generation of IL-2, and probably an intrinsic defect in the responsiveness of MRL-lpr CTLP to IL-2 was contributing to the defective CTL response. We were not able to link suppressor T cells with reduced responsiveness to IL-2. Ageing involves different patterns of change in immunoregulatory T-cell subsets in different strains of mice, depending on their genetic constitution. The general implications of this conclusion for prediction of immune dysfunction with age in genetically distinct members of an outbred population are discussed.     

AT-1.1: A thymus-specific alloantigen of chickens

A thymocyte-specific alloantigen, designated AT (avian thymus) –1.1, has been detected in Cornell C strain (CS) and Obese strain (OS) chickens, the latter being a strain derived from CS which develops a spontaneous form of autoimmune thyroiditis (SAT). Antisera specific for this antigen were developed first in a turkey immunized with thymocytes from an OS chicken and, later, in AT-1.1-negative CS chickens immunized with AT-1.1-positive thymocytes. AT-1.1 was detected in 50–70% of cells in a thymus cell suspension, but was not seen on peripheral blood lymphocytes, erythrocytes, or cells from bursa, spleen, kidney, liver, or brain. It was present on thymocytes of chickens at all ages tested, from 1 day to 6 months of age. AT-1.1 was not detected in six chicken lymphoid tumor cell lines tested, and birds expressing it were found to be negative for the presence of Marek's disease viral antigens. Pedigree studies on 287 (OS × CS)F₂ chickens demonstrated that AT-1.1 is expressed in a dominant or codominant manner, and the gene coding for this antigen was not linked to the B (major histocompatibility) complex. The genetics and tissue distributions of AT-1.1 indicate that it differs from thymus cell surface antigens, avian or mammalian, previously described.     

Thymic development in surgically bursectomized embryonic chicken: expression of PCNA, CD3, CD4 and CD8 markers

Little information is available on the functional relationship between bursa and thymus during chicken embryogenesis. We, therefore, investigated embryonic thymuses taken at 17 days in ovo from chickens bursectomized at 68-72 hours, with histological, histochemical (PAS, Alcian blue), and immunoreaction (anti-cytokeratin B, anti-PCNA/cyclin and anti-CD3, CD4 and CD8 antibodies) methods and compared these data with those from normal and sham-operated chickens of the same age. The bursectomized thymuses distinctly differed from normal and sham-operated thymuses: they were smaller, and the cortical zone was thinner and contained fewer epithelial cells and thymocytes. Only few cortical thymocytes were immunoreactive for PCNA, indicating low proliferative rate. More cortical thymocytes as compared with the normal, expressed CD3 on their cell membrane, whereas the thymocytes at the cortical-medullary border expressing anti- CD4 and anti-CD8 antidodies were less numerous than in normal thymus. The medullary zone contained few epithelial clusters made up of fewer cells than medullary clusters in normal chickens. Some cystic formations were enlarged and contained PAS- or Alcian-blue positive amorphous material. All these data suggest that early bursectomy affects both morphological and functional thymic development.     

Thymus dependency of neonatal tolerance induction in the absence of detectable suppressor cells

Neonatal thymectomy prevents tolerance induction with bovine serum albumin (BSA) in Wistar Furth (WF) rats whose thymus-derived (T) cell deficit is reconstituted with adult nonadherent peripheral blood lymphocytes (PBL). Sham-thymectomized (STx) rats given PBL become tolerant. To establish whether the adult T cells become tolerant in STx rats, their carrier-reactivity was studied in a cooperative immune response following challenge with methylated BSA (mBSA). The results indicate that carrier-reactive cells, derived from PBL, do become tolerant of BSA in the presence, but not in the absence, of the thymus. To determine whether thymic function during tolerance induction is mediated by suppressor T cells, attempts were made to replace the thymus with various populations of thymocytes or lymphoid cells from neonatal or adult normal rats or neonatal BSA-injected rats. No cell population tried could substitute for the thymus during tolerance induction. In addition, it was found that BSA-tolerant rats with intact thymi do not contain either nonspecific suppressor cells whose activity can be boosted with mBSA or specific suppressor activity demonstrable on transfer to normal rats. Timed thymectomy experiments showed that the thymus is required for more than 2, but less than 5 to 7 days after tolerogen injection for significant tolerance induction. These results imply that the thymus itself is necessary for tolerance induction in a peripheral T-cell population and that its effect is not mediated by suppressor cells. It is suggested that peripheral T helper cells may periodically recirculate through the thymus, at least in young rats, and become tolerant of antigen complexed with Ia antigens in the thymic epithelium. Such a mechanism may be of great importance in the development of self-recognition.     

Identification of profound peripheral T lymphocyte immunodeficiencies in the spontaneously diabetic BB rat

The BB rat is presently the best available animal model for human insulin dependent diabetes (IDD). Because of the extreme susceptibility of the strain to opportunistic infections and because current studies suggest that they have an autoimmune diathesis, of which IDD is but one result, aspects of the immune system of the BB rat were studied. Severe T lymphopenia was observed in all BB rats, irrespective of sex or the presence of IDD, while numbers of B cells and serum immunoglobulin levels were normal. Both the helper T lymphocyte and cytotoxic/suppressor T lymphocyte subsets, defined by reactions with monoclonal antibodies, were depressed, and an inversion of the helper T cell subset to cytotoxic/suppressor T lymphocyte subset ratio occurred in all BB rats with increasing maturity. Concomitantly, severe impairments of T cell-mediated immune responses were noted. BB rats poorly rejected allografts across both major and minor histocompatibility barriers, and BB splenic or peripheral blood lymphocytes had markedly defective proliferative responses to mitogens and to allogeneic cells in MLC. Irradiated and nonirradiated BB spleen cells did not inhibit WF mitogenic or MLC responses, which suggests that the T cell defect in BB rats is not solely due to increased suppressor activity. Because irradiated WF cells and Con A supernatants did not restore BB proliferative responses, and BB lymphocytes were able to produce IL-2 normally, a reduced ability of BB lymphocytes to respond to helper factors such as IL-2 is suggested. In contrast to T lymphocytes from spleen or peripheral blood, BB thymocytes responded as well as did WF thymocytes to Con A or Con A supernatants. Percentages of T lymphocyte subsets and histology of BB thymuses were also normal when compared to WF thymuses. However, spleens and lymph nodes from BB rats were severely depleted of T lymphocytes, and thymocytotoxic autoantibodies were detected in many BB rat sera. The above findings indicate that BB rats have T lymphocyte immunoincompetence, which appears to be a post-thymic or peripherally acquired maturational defect.     

Suppressor T cells induced by soluble immune complexes can adoptively transfer inhibition of cytophilic antibody receptors on macrophages.

Immune complexes (soluble antigens of L1210 and antibody to L1210) when given to allogeneic C3H mice generated suppressor cells that inhibited receptors for cytophilic antibody on macrophages. Thymocytes or nylon-nonadherent splenic T cells (4 × 10⁷) from immune-complex-treated mice transferred this suppressive activity when injected into normal syngeneic mice. Maximal suppression of macrophages occurred 4 to 6 days after transfer. In contrast, even 5 × 10⁷ nylon-adherent, non-T spleen cells from immune-complex-treated (“suppressed”) mice failed to induce macrophage suppression in the syngeneic recipients. When T-cell-depleted “B” mice were used as recipients, neither thymocytes nor splenic T cells from suppressed mice were able to transfer suppressive activity. However, the admixture of 2 × 10⁷ normal syngeneic thymocytes with 4 × 10⁷ thymocytes from suppressed mice restored the latter's ability to elicit suppression of macrophages in T-cell-deprived recipients. Peritoneal monocytes from recipients of suppressor thymocytes (to L1210) could not attach cytophilic antibody to L1210 but could attach cytophilic antibody to EL-4 and sheep erythrocytes. Thus, suppressor T cells induced by immune complexes can transfer immunologically specific macrophage suppression (inhibition of cytophilic antibody receptors) to syngeneic recipients. The suppressor cells required the cooperation of normal T cells, suggesting either recruitment of suppressor cells from, or a helper effect by, the normal T cells, in order to produce their effect.     

Tolerance to cyclosporin A-induced autologous graft-versus-host disease is mediated by a CD4+CD25+ subset of recent thymic emigrants

Our previous studies revealed that both the autoeffector and immunoregulatory T cells in cyclosporin A (CSA)-induced autologous graft-vs-host disease are recent thymic emigrants (RTEs). The autoeffector cells appear in and are released from the thymus during the first week of CSA treatment, whereas the immunoregulatory thymocytes appear during the second week but are not released until several days after cessation of CSA treatment. In the present study, the antigenic phenotypes of these functional T cell subsets were determined by immunomagnetic separation and flow immunocytometric analysis. During CSA wk 1, the autoeffector T cells in both the thymus and lymph node (LN) expressed a CD4+8+ double-positive (DP) phenotype, after which those in the LN became CD8 single positive (SP). Timed thymectomy experiments confirmed that the CD8-SP autoeffector T cells in LN originated from these DP RTEs. During CSA wk 2, the immunoregulatory thymocytes also displayed a DP phenotype. However, they were not exported to the periphery until several days after CSA treatment had been interrupted and they had acquired a CD4-SP phenotype. In LN, these immunoregulatory RTEs expressed the CD25+ marker characteristic of anergic/suppressor T cells. Cell separation and mixing experiments demonstrated that the autoeffector T cells persist in LN after cessation of CSA treatment, but their activity is not detectable in the presence of recently exported CD4+ T cells. Hence, the results indicate that tolerance to CSA-induced autologous graft-vs-host disease is actively mediated by CD25+CD4+ RTEs that suppress the function of CD8 autoeffector T cells.     

Helper factor production in murine secondary syngeneic mixed leukocyte reactions

Culture supernatants of murine thymocytes or spleen cells responding in a secondary syngeneic mixed leukocyte reaction (SMLR) were studied for their biologic effects on cell-mediated immune responses in vitro. Such supernatants contained helper factor(s) that facilitated the development of alloantigen-specific cytotoxic T lymphocyte (CTL) responses from thymocyte precursors. Thymocytes, but not spleen cells, required activation by allogeneic effect factor (AEF) in primary culture in order to proliferate and produce biologically active mediator(s) during a secondary SMLR. The same culture supernatants possessed, in some instances, weak T cell growth factor (TCGF; IL 2) activity. However, TCGF activity could be dissociated from helper factor(s) active in the CTL induction assay because some culture supernatants that had potent helper activity were devoid of TCGF activity. This lack of TCGF activity was not due to a lower degree of sensitivity of the TCGF assay or to the presence of a selective TCGF inhibitor in the SMLR-derived supernatants, indicating that the helper factor(s) studied is distinct from TCGF. Production of immunoregulatory lymphokines during the SMLR may serve as a physiologically relevant model for studying the role of T cell-derived lymphokines in immunoregulation.     

An in vitro study of functional maturation of murine thymus cells.

Critical time of onset of thymus cell functions in ontogeny was studied in vitro. Collaborative function in an antibody response and ability to induce a graft-versus-host (GvH) response by murine thymocytes from different stages of ontogeny were investigated. Thymocytes from as early as 16-day mouse embryos were capable of collaborating in the antibody response to sheep-erythrocyte-antigen in vitro following 24 h of pretreatment with concanavalin A (con A). By contrast, maturation of thymus cell function as measured by competence to induce a graft-versus-host reaction, was first manifested by newborn thymus cells, and pretreatment with con A did not facilitate the maturation of this thymus cell function. Experiments to understand the effect of con A on the expression of cell surface antigens have also been reported. Con A-treated thymus cells of different ontogenic stages tested were less susceptible to killing by anti-theta serum than nontreated thymus cells; reverse was true with anti-H-2 serum. The significance of the differential susceptibility of con A-treated thymus cells to anti-sera treatment and the finding that mouse thymocytes can provide helper function as early as the 16th day of gestation have been discussed.     

An in vitro Study of Functional Maturation of Murine Thymus Cells

Critical time of onset of thymus cell functions in ontogeny was studied in vitro. Collaborative function in an antibody response and ability to induce a graft-versus-host (GvH) response by murine thymocytes from different stages of ontogeny were investigated. Thymocytes from as early as 16-day mouse embryos were capable of collaborating in the antibody response to sheep-erythrocyte-antigen in vitro following 24 h of pretreatment with concanavalin A (con A). By contrast, maturation of thymus cell function as measured by competence to induce a graft-versus-host reaction, was first manifested by newborn thymus cells, and pretreatment with con A did not facilitate the maturation of this thymus cell function. Experiments to understand the effect of con A on the expression of cell surface antigens have also been reported. Con A-treated thymus cells of different ontogenic stages tested were less susceptible to killing by anti-θ serum than nontreated thymus cells; reverse was true with anti-H-2 serum. The significance of the differential susceptibility of con A-treated thymus cells to anti-sera treatment and the finding that mouse thymocytes can provide helper function as early as the 16th day of gestation have been discussed.     

Abnormalities of immunoregulatory T cell subsets in patients with insulin-dependent diabetes mellitus

Patients with insulin-dependent diabetes mellitus (IDDM) have autoantibodies that react with cells in the islets of Langerhans. To determine whether these patients suffer from a more generalized immunoregulatory disorder, the ratio of phenotypic helper to suppressor cells was evaluated by specific monoclonal antibodies. Our experiments showed that the helper/suppressor cell ratio was significantly increased in patients with IDDM of less of 2 mo duration and then gradually returned to normal. Despite the alteration in the helper/suppressor cell ratio, there was no evidence for polyclonal activation as measured by the number of immunoglobulin-secreting plaque-forming cells in the peripheral blood. There was, however, a significant increase in the number of spontaneous plaque-forming cells in patients suffering from both IDDM and Hashimoto's thyroiditis (HT). Nonetheless, immunoglobulin production after stimulation with pokeweed mitogen was not different in diabetics with or without HT when compared to normal controls. These findings suggest that subtle changes in the immunoregulatory system occur during the early stages of IDDM.     

Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance

This study shows that the normal thymus produces immunoregulatory CD25+4+8- thymocytes capable of controlling self-reactive T cells. Transfer of thymocyte suspensions depleted of CD25+4+8- thymocytes, which constitute approximately 5% of steroid-resistant mature CD4+8- thymocytes in normal naive mice, produces various autoimmune diseases in syngeneic athymic nude mice. These CD25+4+8- thymocytes are nonproliferative (anergic) to TCR stimulation in vitro, but potently suppress the proliferation of other CD4+8- or CD4-8+ thymocytes; breakage of their anergic state in vitro by high doses of IL-2 or anti-CD28 Ab simultaneously abrogates their suppressive activity; and transfer of such suppression-abrogated thymocyte suspensions produces autoimmune disease in nude mice. These immunoregulatory CD25+4+8- thymocytes/T cells are functionally distinct from activated CD25+4+ T cells derived from CD25-4+ thymocytes/T cells in that the latter scarcely exhibits suppressive activity in vitro, although both CD25+4+ populations express a similar profile of cell surface markers. Furthermore, the CD25+4+8- thymocytes appear to acquire their anergic and suppressive property through the thymic selection process, since TCR transgenic mice develop similar anergic/suppressive CD25+4+8- thymocytes and CD25+4+ T cells that predominantly express TCRs utilizing endogenous alpha-chains, but RAG-2-deficient TCR transgenic mice do not. These results taken together indicate that anergic/suppressive CD25+4+8- thymocytes and peripheral T cells in normal naive mice may constitute a common T cell lineage functionally and developmentally distinct from other T cells, and that production of this unique immunoregulatory T cell population can be another key function of the thymus in maintaining immunologic self-tolerance.     

Further characterization of the suppressor cells, activated by goat anti-Th-B antibody reagent and responsible for enhanced growth of sarcoma 180 in AKR mice.

In our previous study, thymus cells were shown to be responsible for enhancing the growth of the allogeneic sarcoma 180 (S180) in AKR mice that had been injected with goat anti-Th-B antibody reagent (antiserum raised in goats against Balb/c myeloma MOPC 104E cells and purified). We suggested that the cells producing enhancement are suppressor T cells. We now show that the cells responsible for tumor enhancement are indeed T cells, since they carry the Thy-1 antigen on their surface. Treatment of the cells in vitro with anti-Thy-1 plus complement completely eliminates their ability to enhance tumor growth. The thymocytes responsible for tumor enhancement do not carry the Th-B determinant. Treating thymocytes in vitro with goat anti-Th-B antibody reagent plus complement does not abrogate their tumor-enhancing activity. This suggests that the suppressor T cells involved in tumor enhancement are generated by the interaction of anti-Th-B antibodies with precursor suppressor cells which do carry Th-B. Once generated, the active suppressor cells lose the Th-B antigen. This suggestion is supported by our finding that the thymic precursors of Con A-inducible suppressor cells bear Th-B, since they are killed by anti-Th-B plus complement, whereas active suppressor cells induced by Con A do not carry Th-B, since they are not killed by anti-Th-B plus complement. Neither splenic precursors of Con A-inducible suppressor cells nor the active suppressor cells thus induced carry Th-B since neither is killed by anti-Th-B plus complement. We have also found that there are apparently nonthymic suppressor cell precursors which can also be activated by anti-Th-B, since spleen cells from thymectomized mice bearing S180 and treated with anti-Th-B can transfer the tumor-enhancing effect. We conclude that precursors of suppressor cells carry the Th-B determinant. These precursors differentiate to active suppressor cells when stimulated by anti-Th-B antibodies. This process can take place either outside the thymus or in the thymus. Once differentiated, the mature suppressor cells no longer bear the Th-B marker and migrate from their sites of induction. Such cells can suppress immune mechanisms responsible for allogeneic tumor graft rejection and thus cause tumor enhancement.     

Physical separation of "suppressor" from "helper" thymocytes.

Thymocytes from 1-month-old NZB/W mice were separated at unit gravity and then studied for helper or suppressor effects in a GVH assay. Thymocytes with a settling rate of 4 to 6 mm/hr suppressed but did not help. In contrast, help but no suppression characterized thymocytes settling at 6 to 10 mm/hr. Thus helper and suppressor thymocytes in this GVH assay were physically separated.     

Cyclosporine-induced transplantation unresponsiveness in rat cardiac allograft recipients: in vitro determination of helper and suppressor activity

Lymphocytes from spleen, peripheral blood, thymus, and lymph node of naive rats, nonimmunosuppressed recipients of MHC-incompatible heart grafts, and cyclosporine-treated recipients of MHC-incompatible heart grafts were tested for their ability to augment or suppress proliferation of naive cells in an in vitro MLR co-culture assay. Rats treated with cyclosporine for only 7 days maintained their grafts indefinitely. Potent suppressor activity was found in the peripheral blood and spleen of adult naive rats. In untreated engrafted rats, increased suppressor activity was found 1 wk after transplantation and increased helper activity 2 wk after transplantation. In contrast, subnormal helper and suppressor activity was found in cyclosporine-treated rats 1 wk after transplantation. Subsequently, suppressor activity peaked at 2 to 3 wk and helper activity at 4 wk after transplantation. Beyond 5 wk, the cyclosporine-treated rat was indistinguishable from naive ungrafted rats. Two types of suppressor activity were identified that differed in buoyant density and cyclophosphamide sensitivity. Neither suppressor activity demonstrated antigen specificity. These data suggest that one role of cyclosporine in this rat model is to delay the initial helper mechanisms until generalized suppressor activity is operable. The increased antigen-nonspecific activity is only transient, presumably until the final antigen-specific mechanisms become operative.     

Isolation and characterization of novel suppressor T cell clones from murine fetal thymus

Murine fetal thymus from C57BL/6J (B6) and DBA/2J contains a cell population that suppresses CTL responses to alloantigens. This suppressor cell population was found to exist in high frequency in murine fetal thymus at the 14th day of gestation. The activity of this cell in the thymus declined rapidly with increasing time of gestation, and suppressor activity in the thymus was undetectable by the time of birth. On the other hand, suppressor activity could be detected in organ cultures of 14-day fetal thymus even after the organs were cultured for 14 or 21 days. Fetal thymocytes from B6 or DBA/2J mice were grown as long-term lines in interleukin 2 (IL 2)-containing medium. Clones of suppressor cells were derived from long-term cultures by micromanipulation. The clones had an average doubling time of 13 to 16 hr and were dependent on IL 2 for growth. The clones were 10- to 100-fold more efficient in suppressing CTL responses to alloantigens than day 15 fetal thymocytes. Analyses of cell surface molecules with the use of monoclonal antibodies and conventional anti-H-2 sera by radioactive binding assays showed that cloned suppressor cells from B6 fetal thymus were Thy-1 and Lyt-2+, and expressed little or no L3T4, Lyt-1, H-2K, H-2D, and class II molecules. The suppressor clones lacked the cytolytic activity of conventional CTL and also served as very poor target cells in CTL-mediated cytolysis. The suppressor function of the cloned cells was radiation-resistant, and this suppression could not be reversed by the addition of excess exogenous IL 2. The cloned cells suppressed CTL responses only when they were added within the first 48 hr of a 5-day culture period. Analyses of the antigen specificity of the suppressor cells showed that they suppressed CTL responses in a nonantigen-specific manner.     

Recognition of Heterologous Cells by Macrophages

The ability of macrophages to recognize homologous and various heterologous cells was studied in mice, rats, and guinea pigs, in terms of the in vitro phagocytosis of non-opsonized viable thymocytes by macrophages. Mouse, rat, and guinea pig macrophages were found to phagocytize actively thymocytes from certain heterologous animals, including chickens. For instance, mouse macrophages displayed conspicuous phagocytic activities against chicken and duck thymocytes, moderate activities against guinea pig and frog thymocytes and weak activities against rat and mouse thymocytes. On the other hand, guinea pig macrophages revealed a different behaviour: they ingested only chicken thymocytes. These observations strongly suggested that mammalian macrophages possess some ability to discriminate homologous from certain heterologous thymocytes. The results, however, did not necessarily support the idea that the degree of phagocytosis is simply related to the phylogenetic distance between the animal species from which thymocytes and macrophages originated, because of the apparent exception in the mode of phagocytosis by guinea pig macrophages. Evidence demonstrating that antibodies are not involved in this phenomenon will be presented in the accompanying paper.     

Transfer of agammaglobulinemia in the chicken. I. Generation of suppressor activity by injection of bursa cells

Spleen cells from adult agammaglobulinemic (bursectomized) chickens taken 1 to 3 weeks after an injection of histocompatible bursa cells can inhibit the adoptive antibody response to B. abortus of normal spleen or bursa cells in irradiated recipients. Spleen cells from Aγ chickens not injected with bursa cells generally do not. Moreover, bursectomized chickens which have been reconstituted with spleen cells within the first week after hatching do not respond with suppressor cell formation upon bursa cell injection. This apparent “autoimmunization” with bursa cells induces suppressor T cells which are only minimally sensitive to treatment with mitomycin C or to 5000 R γ irradiation. The suppressor activity is neither induced nor potentiated by concanavalin A in vivo. It is much stronger in spleen than in thymus cells and appears to be macrophage independent and to require intact cells. The cell component which stimulates the suppressor activity is more pronounced on bursa than on spleen cells, and is at most present to a very limited extent on bone marrow, thymus, or peritoneal exudate cells. It is better represented in comparable cell numbers of Day 17 than of Day 14 embryonic bursa. The inducing cell component is present in the membrane fraction of disrupted bursa cells. Immunization with bursa cells from B locus histoincompatible chickens leads to suppressor activity against histocompatible bursa cells. Although the removal of Ig-bearing cells from bursa greatly diminishes its immunizing capacity, injection of serum IgM and IgG does not induce suppressor cells. It is suggested that tolerance to a B-cell antigen is lacking in adult Aγ chickens, resulting in an autoimmune response upon exposure to B cells. The B-cell antigen may be a cell surface-specific form of Ig, a complex of Ig and a membrane component, or a differentiation antigen which appears simultaneously with Ig during ontogeny.     

Isolation and characterization of chicken thymic electrolectin.

We have detected the presence of a beta-D-galactoside-binding lectin (electrolectin) in extracts of the thymus of adult chickens. This lectin was purified by affinity chromatography on a lactosyl-Sepharose column to yield 1.4 mg of pure protein from 230 g of thymus. The chicken thymic electrolectin (CTE) has an Mr of 15 300 when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and of 30 000 when analysed by gel filtration. The amino acid composition of CTE is similar to that of other electrolectins purified from human and rat lung. CTE cross-reacts immunologically, but is not identical, with electrolectins from electric-eel electric organ and from chick-embryo pectoral muscle. CTE agglutinates chicken thymocytes but does not appear to promote their mitosis.