Responsiveness of Thymus Cells to Syngeneic and Allogeneic Lymphoid Cells

THE thymus is necessary for the normal development of cell-mediated immunity in mice as shown by the immunological defects after neonatal thymectomy¹. Thymus cells themselves can be stimulated by allogeneic lymphoid cells in mixed leucocyte reaction (MLR)² and become killer cells or cytotoxic lymphocytes after stimulation with allogeneic spleen cells in vitro (H. Wagner and M. Feldmann, unpublished work) and in vivo^3,4. This suggests that the thymus as well as peripheral lymphoid tissues contain T cells which can be stimulated by foreign histocompatibility antigen to divide and differentiate into the cytotoxic lymphocytes which mediate cellular immunity. There have been suggestions that thymus cells might be stimulated to divide by “self” antigen, as well as foreign cells: incorporation of ³H-thymidine above background levels has been found in cultures with syngeneic spleen and thymus cells of adult rats⁵, although the experiments do not determine whether thymus or spleen cells have been stimulated. In contrast to these experiments, Howe et al. reported that only thymus cells of neonatal CBA mice reacted to allogeneic and syngeneic spleen cells of adult animals in “one way” MLR cultures^6,7. Whether the reaction of neonatal thymus cells to syngeneic adult spleen cells is recognition of “self” antigens is uncertain, since spleens of adult mice could carry antigens which do not occur in neonatal animals and are therefore “unknown” for neonatal thymus cells. We demonstrate here that neonatal thymus cells do not react to 4-day-old CBA spleen cells, but adult thymus cells do react against both allogeneic and syngeneic adult spleen 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.     

Isogeneic lymphocyte interactions (ILI) in CBA/N mice: Presence of ILI-Type 2 and absence of ILI-Type 1

The ability of spleen cells from immune defective CBA/N mice to stimulate two types of isogeneic lymphocyte interactions (ILI) was studied. In normal mice adult splenic cells induce proliferation of syngeneic neonatal thymocytes in Type 1 ILI and of syngeneic adult lymph node cells in Type 2 ILI. We have shown that CBA/N spleen cells are inoperative in ILI-Type 1 because the stimulating antigen, murine differentiation antigen 1 (MDA-1), is absent. Murine differentiation antigen 2 (MDA-2), however, is present and Type 2 ILI is evoked by CBA/N spleen cells. The results suggest that MDA-1 and MDA-2 are present on different subsets of B cells. In previous studies in other mice we have shown MDA-1 to be a strong contender for the role of receptor for T-cell signals. Our present findings lend further support to that hypothesis.     

Recovery of the in vitro reactivity of splenic cells of CBA/N mice toward type 2 thymus-independent antigens

CBA/N mice bearing a chromosome X linked immunological deficiency (Xid) cannot respond to type 2 thymus independent antigens (TI-2). However, when their spleen cells are in vitro simultaneously stimulated by both a TI-2 (Fluorescein conjugated polyacrylamide, Flu-PAA) antigen and a type 1 thymus independent (Trinitrophenyl conjugated Brucella abortus, TNP-BA) antigen, their capacity to respond to the TI-2 antigen is recovered. On the contrary, thymus dependent (TD) Sheep red blood cells (SRBC) antigen did not produce any significant increase of the anti-TI-2 response.     

Effect of highly purified thymus factor on the cellular and humoral indices of immunity in thymectomized mice]

Effect of homogeneous polypeptide thymus factor of mol weight about 5000 (thymarin-III) on cellular and humoral immune responses of thymectomized adult CBA mice was studied. Thymectomy proved to greatly decrease the number of T-cells in the spleen. Accordingly, the capability of these mice to produce both IgM and IgG antibody-forming cells in response to the thymus-dependent antigen (sheep red blood cells) was significantly depressed. Subcutaneous injections of thymarin-III (1 microgram per g of body weight) for 7 days completely restored the T-cell spleen population and normalized the animals' immune response.     

Activation of T and B thymus cells to recognize histocompatibility antigens

Lethally irradiated (A × CBA) F₁ or (A × C57BL/6) F₁ mice were injected with 10⁷ A strain thymus cells in attempts to activate donor cells to recognize CBA or C57BL/6 histocompatibility antigens, respectively. Activation could be revealed by injecting activated thymus cells (day 5 irradiated F₁ hybrid spleen cells) into corresponding unirradiated F₁ hybrid hosts. The alloantibody titers formed by these cells and the antirecognition structure (anti-RS) antibody titers induced by them were similar to those observed after injection of normal parental strain spleen cells, indicating that thymus cells had become endowed with recognition structures (RS). Alloantibodies, but no anti-RS antibodies, were present in the serum of F₁ mice given activated thymus cells treated with anti-θ and complement. It, therefore, appeared that activated thymus cells contained sufficient B cells differentiated into antibody-forming cells to give a measurable alloantibody response. On the other hand, receptors responsible for anti-RS antibody induction presumably were located on T cells. Specificity and restriction of antigenic recognition were revealed by negative results obtained when activated thymus cells were injected into F₁ hosts not containing the antigens against which activation had been directed.     

T-cell responsiveness in Mycobacterium lepraemurium infections in a "resistant" (CBA) and a "susceptible" (BALB/c) mouse strain

Antigen-specific and mitogen-nonspecific T-lymphocyte proliferation and lymphokine release (interleukin 2 and macrophage activation factor) were studied in BALB/c and CBA mice infected intravenously with 10(8) Mycobacterium lepraemurium organisms. The responsiveness of spleen cells from infected animals to Con A and specific MLM antigen declined as the infection progressed. Thus, the decreased responsiveness appeared earlier and was more profound in the relatively susceptible BALB/c strain than in the relatively resistant CBA strain. Nylon-wool-purified, T-cell-enriched spleen cells from both strains, however, responded to both M. lepraemurium antigen and Con A until the later stages of infection (17 weeks postinfection). The relevance of nonspecific immunodepression mediated by nylon-wool-adherent spleen cells to the progressive nature of this infection is discussed.     

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.     

Induction of tolerance against the Mls antigen in mice no need for Mls-bearing cells

Infusion of CBA mice with spleen cells from the H-2-compatible, but Mis antigen-incompatible, strain C3H leads to a specific reduction of the MLC reactivity of the host's lymphocytes. One explanation of the reduced reactivity could be that the specifically Mls antigen-responsive CBA T cells become exhausted by intense antigen stimulation or that the infused cells actively neutralize specifically responsive cells. In this investigation, we have shown that depletion of membrane Ig-positive cells from C3H × CBA spleen cell preparations strongly reduces their capacity to stimulate CBA lymphocytes in the MLC, indicating that the Mls antigen is expressed on B but not on T cells. However, such B-cell depleted cell preparations were fully capable of reducing the MLC response of CBA hosts. Cell preparations of spleen and lymph nodes exhibited high stimulatory and inhibitory effects. Thymic cells lacked both these characteristics, whereas bone marrow cells were weak stimulators but relatively strong inhibitors. The results support the proposal that the observed reduction of MLC reactivity is due to an active process of the injected cells. The cell type which is working as an inhibitor has not been clearly defined yet.     

In vitro studies of the genetically determined unresponsiveness to thymus-independent antigens in CBA/N mice.

The X-chromosome-linked B lymphocyte defect of CBA/N mice has been studied in vitro by comparing the ability of (CBA/N X DBA/2)F1 (X-/X- X X+/Y) male (X-/Y) and female (X-/X+) spleen cells to respond to the thymus-independent antigen DNP (or TNP)-AECM-Ficoll. (CBA/N X DBA/2)F1 male spleen cells failed to generate significant in vitro anti-TNP antibody responses to DNP- or TNP-AECM-Ficoll, in contrast to spleen cells from F1 female (X-/X+) mice which responded normally to these T-independent antigens. Spleen cells from male F1 mice responded almost as well as F1 female cells to the thymus-dependent antigen, TNP-sheep red blood cells (TNP-SRBC) in vitro. Adding F1 male cells to F1 female cells failed to reduce the response of the latter to DNP-AECM-Ficoll, suggesting that the inability of F1 male cells to respond was not due to active suppression. The response of F1 male spleen cells to TNP-SRBC was not impaired by adding high concentrations of TNP-AECM-Ficoll indicating that the mechanism of unresponsiveness was not tolerance induction in all TNP-specific precursors. Lymphocytes from F1 male mice were capable of forming anti-TNP antibody after stimulation with lipopolysaccharide (LPS) in high concentrations; DNP-AECM-Ficoll had no effect on this polyclonal response. B lymphocytes from mice bearing only the X-chromosome of the CBA/N strain thus display a profound defect in B cell activation. This functional defect may represent either an inability of the defective B cells to be activated by thymus-independent antigens or the absence of a sub-class of B cells which respond to thymus-independent antigens.     

Monoclonal antibodies as probes for differentiation and tumor-associated antigens: a Forssman specificity on teratocarcinoma stem cells

A set of monoclonal antibodies derived by fusing P3-NS1/1-Ag4-1 myeloma cells with spleen cells from a rat immunized with mouse spleen were screened for activity against a tumor cell panel. One of these antibodies was found to react only with mouse embryonal carcinoma cells and no other tumor cell type tested, including differentiated derivatives of teratocarcinomas. In the adult mouse, this antigen is expressed by subpopulations of cells in the spleen, bone marrow, lymph node, brain, kidney and testes, although not in liver and thymus. This antigen has a species and tissue distribution consistent with that of Forssman antigen. The molecules which carry this specificity on the embryonal carcinoma cells appear to be glycolipids.     

Distribution of cells bearing the Tac antigen during ontogeny of human lymphoid tissue

The monoclonal antibody anti-Tac, which binds to the interleukin 2 (IL 2) receptor, was used to identify this antigen in human fetal and adult lymphoid tissue. Liver, spleen, thymus, lymph node, and peripheral blood were examined for Tac-positive cells with the use of frozen sections or cytocentrifuge preparations. The results show that cells in the fetal and neonatal thymus express the Tac antigen; these cells are predominantly located in the medulla. The liver and spleen of both fetus and adult exhibit very few Tac-positive cells. Double staining demonstrates that cells bearing the Tac-antigen stain with Leu-4, an anti-T cell antibody. In adult lymph node tissue, the Tac-bearing cells are predominantly distributed in the interfollicular area, with positive cells also present in the germinal center and mantle zone. The Tac antigen is present on both T and B cells. Few Tac-positive cells are present in the circulating peripheral blood.     

The origin of antibody-forming cells detected in the bone marrow after thymus-independent antigen-stimulation and abnormality in migration of B cells of X-linked immunodeficient CBA/N mice

The anti-TNP IgM plaque-forming cells (PFC) were generated in the spleen and bone marrow of non-immunodeficient normal mice after intraperitoneal administration of TNP-LPS. Irradiation of normal mice while shielding bone marrow completely abrogated the generation of bone marrow PFC, indicating that they are derived from extramedullary sites. The bone marrow PFC, response to TNP-LPS was low in X-linked immunodeficient CBA/N strain mice, while the spleen response was comparable to that seen in the normal mice. To further study the basis of the deficient bone marrow PFC response in CBA/N mice, spleen cells were adoptively transferred to irradiated syngeneic mice stimulated with TNP-LPS. While spleen cells from normal mice generated high numbers of PFC in recipient bone marrow and spleen, those from CBA/N strain mice could not generate bone marrow PFC. This result was obtained regardless of whether normal or CBA/N recipients were used. These results indicate that TNP-LPS administration normally results in the migration of B lymphocytes from the periphery into the bone marrow and that B cells from immunodeficient CBA/N strain mice bear an inherent defect in this migratory function. This migratory defect was shown to be X-linked, as are the other previously reported B cell defects in this inbred mouse strain. The possible relationship between this migratory defect and the maturational defects of B cell lineage as reported previously in CBA/N strain mice is discussed.     

INFLUENCE OF AGE AND ANTIGENIC STIMULATION ON GRANULOCYTE AND MACROPHAGE PROGENITOR CELLS IN THE MOUSE SPLEEN

The frequency and proliferative activity of granulocytic and macrophage progenitor cells were determined in the spleens of C₅₇BL, BALD/c, NZB and CBA mice. These cells were detected by their capacity to form granulocytic and/or macrophage colonies ( in vitro colony-forming cells, CFC) in agar culture. In vitro CFCs were low in frequency in the adult spleen (4–28/10⁵ cells) compared with the bone marrow (180–280/10⁵ cells). However, the neonatal spleen, both in germfree and conventional mice, contained high levels of in vitro CFCs. From the low suiciding index with tritiated thymidine and the small numbers of cluster-forming cells in relation to colony numbers, many in vitro CFCs in the adult C₅₇BL spleen appear to be in a non-cycling state. The level and activity of in vitro CFCs were extremely low in the spleen of adult germfree CBA mice but were greatly increased in conventional mice following the injection of a bacterial antigen.     

Absence of suppression in natural and induced tolerance to F antigen

The role of suppression in natural and induced tolerance to F antigen was investigated in two sets of experiments. In the first, CBA mice were submitted to pretreatments which decrease suppression and the antibody response to self- or allo-F type was investigated. The second set of experiments involved the transfer of spleen cells from tolerized or from naturally tolerant mice into normal mice which were then primed with allo-F, as well as the co-transfer of tolerant and primed lymphocytes into normal mice, to test whether tolerant lymphocytes present suppressor cells. The results indicate that the immune response against allo-F antigen is normally kept in a low level by a suppressive mechanism, and that F-specific suppressor T cells are absent from tolerant mice.Abbreviations used in this paper ATx adult thymectomy - BSS buffered salt solution - CFA Freund's complete adjuvant - CY cyclophosphamide - F.1 type-1 F antigen - F.2 type-2 F antigen - PBS phosphate-buffered saline - RIA radioimmunoassay - Th T helper cell - Ts T suppressor cell     

The enrichment of thymocytes bearing C3 receptors following cortisone involution.

Young CBA mice were injected with 2.5 mg of cortisone acetate, following which their spleen cells, thymocytes, and lymph node cells were tested for receptors for the third component of complement (C3) over a 20-day period. An erythrocyte-antibody-complement (EAC) rosette assay was used to detect C3 receptors. Cells bearing C3 receptors in the thymus emerged as early as 2 days after cortisone injection and peaked to a level of 18% at Day 7. This was followed by a decline to control levels by Day 14. There was no significant change in the percentage of C3 receptor-bearing cells in lymph node and spleen of the cortisone injected animals compared to appropriately matched uninjected animals. Evidence has been presented that cortisone-resistant thymocytes may bind EAC, and that these cells are surface Ig negative, contain no or very few macrophages, and bear thy-1 antigen. Complement receptor lymphocytes (CRLs) were separated from the nonrosetting thymus cells by sedimentation in an Isopaque-Ficoll gradient. These enriched C3 receptor-bearing cells were found to possess thy-1 antigens by indirect immunofluorescence. Specificity controls using antisera with thy-1.1 and thy-1.2 specificity and donor animals with thy-1.1 or thy-1.2 phenotypic expression indicated that C3 receptor-bearing cells appearing in the thymi of these respective donors following cortisone involution possessed the appropriate thy-1 phenotype.     

T cell antigen receptor expression by subsets of Ly-2-L3T4- (CD8-CD4-) thymocytes

The V beta 8-specific mAb F23.1 and KJ16 were used as fluorescent stains to test for TCR expression on the surface of subpopulations of early, CD4-CD8- (L3T4-Ly-2-) thymocytes from adult CBA mice. A surprisingly high proportion (27%) of Ly-2-L3T4- thymocytes were strongly F23.1 and KJ16 positive. No positive cells were detected among Ly-2-L3T4- thymocytes from V beta 8-negative SJL mice. In contrast to the adult thymus, Ly-2-L3T4- cells from embryonic CBA thymus lacked F23.1-positive cells. Subsets of adult CBA Ly-2-L3T4- thymocytes were separated to determine which expressed V beta 8. The major subset, Ly-1 low B2A2-M1/69+Thy-1+Pgp-1-, representing a phenotype similar to embryonic Ly-2-L3T4- thymocytes and the phenotype commonly isolated from adult thymocytes as Ly-1 "dull," lacked cells strongly positive for F23.1. In contrast, a series of subsets of adult CBA Ly-2-L3T4- thymocytes which were B2A2-M1/69- and Pgp-1+ all included strongly F23.1-positive cells. A minor subset, negative for most markers except Pgp-1 and presumed on the basis of this phenotype and some reconstitution studies to include the earliest intrathymic precursors, contained 28% F23.1-positive cells. However, no F.23.1-positive cells were detected in equivalent "prethymic" populations from bone marrow or from athymic mouse spleen. The subsets of Ly-2-L3T4- thymocytes which were Ly-1 high, B2A2-M1/69-, and Pgp-1+ all contained about 70% F23.1-positive cells, indicating a V beta 8 usage much higher than the mature T cell average. These results indicate that a series of distinct developmental events have occurred within these CD4-CD8- thymocytes previously considered as a single group of early precursor cells, and that some aspects of repertoire selection may be occurring amongst thymocytes which lack CD4 or CD8.     

B-cell suppression of antibody response to sheep red blood cells in mice of high- and low-responding genotypes.

CBA and C57B1 mice (high and low responders to sheep red blood cells, respectively) were injected intravenously with syngeneic lymph node, marrow, spleen, or thymus cells together with sheep red blood cells (SRBC), and the production of antibody-forming cells (AFC) was assayed in the spleen. Transfer of lymph node, marrow, spleen, or thymus cells led to a significant enhancement of immune responsiveness in low-responding C57B1 mice. In contrast, transfer of marrow, lymph node, or spleen cells to high-responding CBA mice was accompanied by a decline in AFC production. These effects were magnified if syngeneic cell donors had been primed with SRBC; suppression in CBA mice and stimulation in C57B1 mice were especially pronounced after transfer of SRBC-primed lymphoid cells. Pretreatment of CBA donors with cyclophosphamide in a dose causing selective B-cell depletion completely abrogated the suppression of immune responsiveness. A large dose (10⁷) of syngeneic B cells injected together with SRBC suppressed the accumulation of AFC in both CBA and C57B1 mice. No suppression of immune responsiveness was observed after transfer of intact thymus cells, hydrocortisone-resistant thymocytes, or activated T cells. We conclude that suppression of the immune response to SRBC is induced by B cells. At the same time, there is a possibility that the addition of “excess” B cells acts as a signal, triggering suppressor T cells.     

Cell transfer studies in cyclophosphamide-induced tolerance

Thymectomized, irradiated adult CBA mice were restored with various combinations of bone marrow and thymus cells from nontolerant animals and from animals made tolerant to sheep erythrocytes or to hemocyanin with the drug cyclophosphamide. Mice reconstituted with tolerant marrow and thymus responded as well as those that received nontolerant cells. Thus it is concluded that the tolerant state of the transferred marrow and thymus cells is not a significant factor in the tolerant state of the recipient, and that antigenic diversity is restored in the interaction and proliferation of bone marrow and thymus cells that follow transfer.Thymectomized irradiated mice restored with thymocytes, in contrast to unoperated animals, require multiple antigen injections to demonstrate comparable immune response, but develop tolerance normally when treated with cyclophosphamide and antigen. Reconstitution with tolerant marrow and thymus cells resembles the recovery of immune responsiveness seen after lethal irradiation of tolerant mice; in both instances a complete breakdown of immunological tolerance is observed.     

Colony formation in agar by adult bone marrow multipotential hemopoietic cells

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen-stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 10⁵ bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony-forming cells. In CBA mice erythroid colony-forming cells were mainly non-cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H-thymidine). Erythroid colony-forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU-S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony-forming cells and CFU-S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine-positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU-S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony-forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.