Factor producing lysis of thymus-derived cells in vitro by specific antigen.

Lymph node cells of immunized mice were lysed by specific antigen in vitro. These cells were not lysed by antigen after washing them four times with Hanks' solution. The factor lysing lymph node cells and thymus cells of normal mice in the presence of the specific antigen was found in the supernatant of the lymph node cell suspension of immunized mice. Bone marrow cells were also lysed by the factor and specific antigen only after preincubating the cells in extracts of thymus or lymph nodes of normal mice. When active supernatants were fractionated by gel filtration on Sephadex G-200, the active material eluted with molecules of approximately 20,000–45,000 MW. This factor was homogeneous on electrophoreses in polyacrylamide gels and detected in the prealbumin zone.     

Enhanced suppression of blastogenic responses by weanling mouse lymphoid cells treated with tetrahydrocannabinol in vitro

The suppressive effects of delta 9-tetrahydrocannabinol (THC) on the proliferation of lymphocytes from the spleen, lymph node, and thymus of weanling animals vs adult animals to the T-cell mitogen PHA were examined. THC had a suppressive effect on thymus cells from animals of both younger and older mice. THC suppressed spleen and lymph node cells responses to phytohemagglutinin (PHA) more readily when the cells were obtained from young mice rather than older animals. Suppression by THC in the adult mice was greater in an organ containing fewer mature T lymphocytes such as the thymus in comparison to lymphocytes in secondary organs such as the spleen and lymph nodes which contain more mature lymphocytes.     

Some endocrine aspects of the thymus gland.

In studies of the mouse thymus, lymphocyte mitoses are seen to be most frequent in the thymus cortex. There is evidence from thymic grafts that a hypothetical factor, thymopoietin, may stimulate mitosis of thymic lymphocytes. It is a factor which is postulated to act in conjunction with the PAS-positive mesenchymal reticular cells and epithelial reticular cells of the cortex. The thymus medulla is necessary for the integrity of thymic grafts, and may also elaborate a secretion for maintaining the cellular functions of the gland. Thymectomy has been used as a gauge for judging normal thymic function and results, in the mouse, in lymphopenia, degeneration of spleen and lymph nodes, delayed rejection of skin allografts, reduced ability of spleen cells to mount the graft versus host reaction, and reduced primary immune response to certain antigens. Correction of these deficiencies offers a means of evaluating various thymic extracts and grafts. Lymphocytosis-stimulating hormone (LSH) is known to maintain the peripheral lymphoid organs and cause lymphocytosis in the thymectomized animal. Diffusion chamber studies of thymic grafts also show restored lymphoid tissue by a cell-free factor (CIF). These two factors may be the same and probably represent the basis of the highly purified lymphocyte-stimulating proteins, LSHr and LSHh, which restore the L/P ratio in thymectomized animals and may stimulate lymphopoiesis in spleen and lymph nodes. LSHr, unlike LSHh, increases the total lymphocyte count. LSHr has been found to increase the humoral antibody response in neonatal mice both by the PFC technique and by direct hemolysis of sheep erythrocytes. Homeostatic thymic hormone (HTH) is a thymic extract of small molecular weight and contains nucleic acid. In the thymectomized guinea pig it has been found to maintain normal levels of lymphocytes in the blood, spleen and lymph nodes, to restore antibody titers to typhoid H antigen and to restore the toxic allergic reaction. Thymic humoral factor (THF) is of smaller molecular weight (less than 1,000) and probably is not a protein. It also enhances lymphoid proliferation in neonatally thymectomized mice. There is evidence that THF participates in humoral antibody formation because it stimulates PFC formation from neonatally thymectomized mice after inoculation with sheep erythrocytes. Its effects on cell-mediated immunity are seen from findings that injection of THF restores the ability of thymectomized mice to reject skin allografts. THF enables spleen cells from thymectomized or neonatal animals to mount the graft versus host reaction, and causes maturation of bone marrow cells and spleen or lymph node cells so that they can participate in the graft versus host reaction. It has been reported to stimulate lymphocytes to kill isogeneic tumor cells in vitro. Thymosin is protein extracted from the thymus. It has been found to alleviate leukopenia slightly and provide some improvement in lymphoid histology in thymectomized mice...     

Contact sensitivity to picryl chloride: the occurrence of B suppressor cells in the lymph nodes and spleen of immunized mice.

Mice were immunized for contact sensitivity and antibody production by painting the skin with picryl chloride. Lymph node and spleen cells taken 4 days later transferred contact sensitivity. However, cells taken at 7–8 days failed to transfer but were able to block the transfer by 4 day immune cells. These suppressor cells occurred in the regional lymph nodes, spleen and thymus. The suppressor activity of lymph node and spleen cells was due to B cells as shown by the effect of anti-θ serum and complement, nylon wool filtration and separation of EAC positive and negative cells by centrifugation on a discontinuous gradient. The transfer of fractions rich or poor in macrophages showed that the suppressor cell in the transferred population was not a macrophage. Separation using EAC rosettes suggested that B cells were responsible for the suppressor activity in the thymus.T cells isolated from the lymph nodes and spleen 7–8 days after immunization transferred contact sensitivity although the initial population was inactive. This indicates that passive transfer cells are present in the regional lymph nodes and spleen at later times after immunization but cannot be demonstrated because of the presence of suppressor B cells. However, no passive transfer cells were found in the thymus. The production of B suppressor cells required little or no T cell help and following immunization the spleens of reconstituted (B) mice were at least as active as control cells in causing suppression. There are several different suppressor cells which act in the picryl system and the B suppressor cells in immunized mice described here are distinct from the T suppressor cells in mice injected with picryl sulphonic acid.     

Changes in the H-alloantigen-recognizing function of mouse lymphoid cells following hydrocortisone administration]

The capacity of spleen, thymus, and bone marrow cells of intact (control) and of hydrocortisone-treated mice CBA to induce the lymph node type of graft-v-host reaction (GVHR) in hybrids F1 (CBA X c57bl) was studied. After hydrocortisone injection (2.5 mg per mouse) the donor spleen cells became more active in GVHR, considering the value of lymph node indices and immunoblast content in the regional lymph node as compared with a control group. Following transplantation of thymus cells taken from the hydrocortison-treated donors the immunoblast count was higher, although the lymph node weight remained the same as in the control group. On the contrary, following the transfer of the bone marrow cells from the hydrocortisone-treated mice the lymph nodes enlarged, while the immunoblast count remained as low as in control. Consequently, exogenously conditioned increase in the hydrocortisone level was accompanied by an enrichment of the spleen and thymus cell populations with T-lymphocytes, proliferating in response to H-alloantigens.     

Studies on the recirculating cells in the mouse thymus

The cells in the mouse thymus which respond to PHA and have the distribution characteristics of recirculating lymphocytes upon infusion into isologous recipients are markedly enriched 2 days after treatment of mice with proper doses of cortisone acetate. However, a similar increase is not observed in lymph nodes and spleen. During regeneration of the steroidinvoluted thymus, the proportion of recirculating thymic cells disappeared more rapidly than the relative PHA-responsiveness of the cell population. These findings indicate that all PHA-responsive thymocytes may not have recirculating capacity. The recirculating cells present in the thymus and lymph nodes have no tendency to distribute to the thymus, but tend to accumulate in lymph nodes. This fact, together with the finding that in vitro trypsin treatment of the cells temporarily abolishes their lymph node-seeking capacity, are taken as evidence that the recirculating cells in the thymus have undergone their maturation within this organ whereby they have gained membrane receptors which determine their distribution characteristics.     

Development of follicular dendritic cells in lymph nodes of B-cell-depleted mice

Summary We have studied follicular dendritic cells (FDC) in lymph nodes of normal and thymus dysgeneic nude mice depleted of B-cells by chronic treatment with anti-IgM antibodies. We found that B cell depletion was accompanied by the absence of mature FDC as defined morphologically at the ultrastructural level. Only precursor FDC (p-FDC) could be demonstrated. Upon release of B-cell suppression, the repopulation of lymph nodes with B-cells was associated with the reappearance of fully differentiated FDC in primary follicles of nude mice and in secondary follicles of T-cell competent mice. We conclude that mature B-cells and/or B-cell products are required for the development of mature follicular dendritic cells in the mouse lymph node.     

Effect of thymus cell injections on germinal center formation in lymphoid tissues of nude (thymusless) mice

Nude mice, partially backcrossed to Balb/c or DBA/2, were injected iv with 5 × 10⁷ thymus cells from the respective inbred strain. The response of these mice to immunization with Brucella abortus antigen was studied, with respect to both antibody production and the formation of germinal centers in their lymphoid tissues. The results were compared to those obtained with nude mice to which no thymus cells were given, as well as to Balb/c, DBA/2, or +/? litter mate controls.Nude mice formed less 19S as well as 7S antibody than did litter mate controls and completely lacked germinal centers in lymph nodes and gut-associated lymphoid tissue. Those nude mice which had been injected with thymus cells made a much better secondary response, both for 19S and for 7S antibody, and had active germinal centers in their lymph nodes as early as 3 wk after thymus cell injection. Intestinal lymphoid tissue in nude mice showed only slight reconstitution of germinal center activity several months after thymus cell injection and none at earlier times. Irradiated (3000 R) thymus cells appeared as effective as normal cells in facilitating germinal center appearance and 7S antibody production in the nude mice.     

The nucleolar apparatus of the lymphoid cells in AKR mice at different stages of leukemogenesis

Morphological features of nuclear apparatus in mice leukemic cells of the peripheral blood, lymph nodes, thymus, bone marrow and spleen were studied. These cells were taken from 12-months-old AKR mice. Enlarged percentage of cells with nucleolar lipid component in thymus characterizes preleukemic state for 6-months-old AKR line mice.     

The role of the thymus in the ontogeny of the immune system

The proper development of the organs of the immune system is dependent on at least three factors: (1) the development of anlagen with the capacity to trap antigens and support the proliferation of lymphoid and plasma cell precursors; (2) the production by the bone marrow of lymphoid and plasma cell precursors which seed in the lymphoid organs; and (3) the thymus, which seeds reactive cells to the lymphoid organs and produces a humoral factor stimulating antigen-triggered proliferation of primitive lymphoid and plasma cells. Studies on cell population changes in the lymph nodes following thymectomy in mice confirm earlier evidence that most cells produced in the thymus do not seed to the lymphoid organs, but die locally in the thymus.     

T cell lineages in the thymus of lpr/lpr mice. Evidence for parallel pathways of normal and abnormal T cell development

Autoimmune mice homozygous for the lpr/lpr (lpr) gene develop a profound lymphadenopathy resulting from the accumulation of immature Thy-1+ Lyt-2- L3T4- cells in peripheral lymphoid tissues. The source of these cells is not known although the presence of a thymus is necessary to manifest both the lymph node enlargement and the autoimmunity. For this reason and the fact that the abnormal lpr cell phenotypically resembles immature thymocytes, we studied the thymus in lpr mice. Adult lpr thymuses were found to contain an immature population phenotypically identical to the peripherally accumulating cells, including the expression of B220 and Pgp-1 antigens as well as the presence of surface T cell receptor molecules as defined by the antibody KJ16-133. Evidence is presented that some of these lpr precursor T cells are capable of intrathymic differentiation, whereas the vast majority are exported unchanged to the lymph nodes where a portion differentiate further into mature T cells. This lpr-specific lineage could be distinguished from a normal component of the lpr thymus by surface phenotype and immunohistology. The results suggest that the massive accumulation of cells in lpr lymph nodes is not so much the result of abnormal proliferation of T cells as abnormal intrathymic differentiation. In addition, a minor subpopulation of normal Lyt-2- L3T4- thymocytes was identified that resembles the phenotype of the lpr cell and similarly expresses surface T cell receptor molecules. The presence of two parallel lineages in the lpr thymus thus also provides insight into normal T cell development.     

The restoration of the T-lymphoid system of nude mice: lower efficiency of nonlymphoid, epithelial thymus grafts.

The T-cell deficiency of nude mice is due to an abnormal differentiation of the thymus epithelium; it can be persistently corrected by grafting a neonatal thymus. However, grafted adult thymuses or epithelial thymuses are not repopulated by large numbers of host-derived lymphocytes, as is the case when a whole neonatal thymus is grafted. Furthermore, the repopulation of the spleen and lymph nodes by T cells is less pronounced than after whole neonatal thymus transplantation, and the restoration of the reactivity to T-cell mitogens is irregular. Therefore, the integrity and the age of the thymus graft are important for a good restoration of the T-lymphoid system of congenitally athymic animals.     

Histochemical localization of autometallographically detectable mercury in tissues of the immune system from mice exposed to mercuric chloride

Summary The distribution of mercury in the spleen, liver, lymph nodes, thymus and bone marrow was studied by autometallography in mice exposed to mercuric chloride intraperitoneally. Application of immunofluorescence histochemistry and an autometallographic silver amplification method was employed to the same tissue section. Mercury was not only detected in macrophages marked by the antibody M1/70 but also in macrophage-like cells, which were either autofluorescent or devoid of fluorescent signals. These two cell types were identified as macrophages at the electron microscopical level. Autometallographically stained macrophages were observed in the spleen, lymph nodes, thymus and in Kupffer cells of the liver. Furthermore, mercury was observed in endothelial cells. No obvious pathological disturbances were observed at light and electron microscopical level. At the subcellular level mercury was localized in lysosomes of macrophages and endothelial cells.     

Pituitary Adenylate Cyclase Activating Peptide Deficient Mice Exhibit Impaired Thymic and Extrathymic Regulatory T Cell Proliferation during EAE

We have shown that mice deficient in pituitary adenylate cyclase-activating polypeptide (PACAP, gene name ADCYAP1) manifest enhanced sensitivity to experimental autoimmune encephalomyelitis (EAE), supporting the anti-inflammatory actions described for this neuropeptide. In addition to an increased proinflammatory cytokine response in these mice, a reduction in regulatory T cell (Treg) abundance in the lymph nodes (LN) was observed, suggesting altered Treg kinetics. In the present study, we compared in PACAP deficient (KO) vs. wild type mice the abundances and rates of proliferation FoxP3⁺ Tregs in three sites, the LN, central nervous system (CNS) and thymus and the relative proportions of Th1, Th2, and Th17 effector subsets in the LN and CNS. Flow cytometry analyses revealed a decrease in Treg proliferation and an increased T effector/Tregs ratio in the LN and CNS of PACAP KO mice. In the thymus, the primary site of do novo natural Treg production, the total numbers and proliferative rates of FoxP3⁺ Tregs were significantly reduced. Moreover, the expression of IL-7, a cytokine implicated in thymic Treg expansion during EAE, failed to increase at the peak of the disease in the thymus and LN of PACAP KO mice. In addition to these Treg alterations, a specific reduction of Th2 cells (about 4-fold) was observed in the lymph nodes in PACAP KO mice, with no effects on Th1 and Th17 subsets, whereas in the CNS, Th1 and Th17 cells were increased and Th2 decreased. Our results suggest that endogenous production of the neuropeptide PACAP protects against EAE by modulating Treg expansion and Th subsets at multiple sites.     

Experimental Trypanosoma cruzi infection alters the shaping of the central and peripheral T-cell repertoire

We investigated the thymic and peripheral T-lymphocyte subsets in BALB/c mice undergoing acute or chronic Trypanosoma cruzi infection, in terms of expression of particular Vbeta rearrangements of the T-cell receptor. We first confirmed the severe depletion of CD4(+)CD8(+) thymocytes following acute T. cruzi infection. By contrast, the numbers of CD4(+)CD8(+) cells in subcutaneous lymph nodes increased up to 16 times. In subcutaneous lymph nodes, we found CD4(+)CD8(+) cells that expressed prohibited segments TCRVbeta5 and TCRVbeta12 (which are physiologically deleted in the thymus of BALB/c mice), as did some mature single-positive cells (CD4(+)CD8(-) and CD4(-)CD8(+)). In the thymus of infected animals, although higher numbers of immature cells bearing such Vbeta segments were seen, they were no longer detected in the mature single-positive stage, suggesting that negative selection occurs normally. We also found increased numbers of cells bearing the potentially autoreactive phenotype TCRVbeta5(+) and TCRVbeta12(+) in T-lymphocyte subsets from subcutaneous lymph nodes of T. cruzi chronically infected mice. In conclusion, our data indicate that immature T lymphocytes bearing prohibited TCRVbeta segments leave the thymus and gain the lymph nodes, where they further differentiate into mature CD4(+) or CD8(+) cells. Conjointly, these findings show changes in the shaping of the central and peripheral T-cell repertoire in both acute and chronic phases of murine T. cruzi infection. The release of potentially autoreactive T cells in the periphery of the immune system may contribute to the autoimmune process found in both murine and human Chagas' disease.     

Immunosuppression and lymphoid hypoplasia associated with chronic graft versus host disease is dependent upon IFN-gamma production

We have previously shown that both IFN-gamma and IFN-beta are produced in vivo and in vitro by spleen cells obtained from mice experiencing a chronic form of graft vs host disease (GVHD). Further, we have shown that in vitro production of IFN-beta by spleen cells from GVHD mice may play a role in the suppressed in vitro mitogen responsiveness of these cells. This study was undertaken to investigate if treatment of such mice with mAb to IFN-gamma or IFN-beta could alter the immunosuppression or lymphoid hypoplasia associated with chronic GVHD. GVHD was induced across minor histocompatibilities by the i.v. injection of B10.D2 spleen cells into sublethally irradiated BALB/c mice. These mice were given daily injections for 20 days of one of the following: 1) mAb to IFN-gamma, 2) mAb to IFN-beta, or 3) control IgG. Histologic examination of these mice at 21 to 22 days post transplantation revealed that mice treated with mAb to IFN-beta or control IgG had dramatic hypoplasia of the thymus, spleen, and lymph nodes which was similar to untreated GVHD mice. Mice given mAb to IFN-gamma, however, had no lymphoid hypoplasia and had a near normal gross and histologic appearance of their thymus, spleen, and lymph node tissue when compared with syngeneic controls. In vitro mitogen-induced proliferative responses of spleen and lymph node cells obtained from GVHD mice or GVHD mice treated with mAb to IFN-beta were severely suppressed or absent. In contrast, spleen and lymph node cells from GVHD mice given mAb to IFN-gamma were capable of giving a significant in vitro proliferative response to Con A, PHA, and LPS. Further, natural suppressor cell activity and spontaneous production of IFN-beta, a characteristic of this form of GVHD, was absent in spleen cells obtained from GVHD mice treated with mAb to IFN-gamma. These results further identify the IFN as playing critical roles in the pathogenesis of GVHD.     

A system of alloantigens that selectively identifies lymph-node lymphocytes

The antiserum (BALB.I-H-2 ^j x SWR/7)F₁ anti-I.29 ascites cells, in reaction with B6 lymph-node cells (LNC) in the cytotoxicity assay, defines an alloantigen system called Lna-1* (lymph-node alloantigen-1) which in normal, untreated mice is expressed on the cells of lymph nodes but not of other lymphoid organs. The T- and B-cell populations of lymph nodes evidently include Lna-1u1) subpopulations representing 30–40 percent of the total population. The Lna-1¹ phenotype could be induced on cells of thymus and spleen but not of bone marrow. Congenitally asplenic +/Dh mice have no Lna-1⁺ cells in their lymph nodes, but their LNC can be induced to express Lna-1; this suggests that the spleen is normally required for the differentiation of Lna-1⁺ cells from Lna-1^– precursors. It is not yet known whether thymus is also required for the expression of Lna-1 in lymph nodes. It remains to be seen whether the existence of the Lna-1 + B-cell subpopulation of lymph nodes depends on Lna-1⁺ T cells, and whether the Lna-1⁺ phenotype of B cells may be acquired rather than intrinsic. One hypothesis which is the basis of further study is that there is a T-cell pathway in which noninducible bone-marrow cells become Lna-l-inducible in the thymus, then travel to the spleen, where they are induced to become Lna-1⁺, after which they reside in lymph nodes.Dr. Jan Klein reminded the authors that Lna was previously used in reference to la antigens (David et al. 1973) and was later abandoned.