Neonatal Thymectomy Research Articles

Defective suppressor function of spleen cells by neonatal thymectomy and persistence of experimental auto‐immune hepatitis in mice

The effects of neonatal thymectomy on a model of auto‐immune hepatitis prepared by immunizing A/J mice with syngeneic crude liver proteins were studied. Hepatitis was more severe in neonatally thymectomized mice (Group B) than in non‐thymectomized controls (Group A). Changes in the hepatic lobules were reduced in both groups 3 months after the final immunization, but inflammation around the portal areas tended to persist in Group B; only this group showed frequent infiltration of mononuclear cells accompanied by destruction of the limiting plate in the portal area. The serum level of auto‐antibody to liver‐specific membrane lipoprotein complex (LSP) and delayed‐type hypersensitivity (DTH) to LSP were higher in Group B than in Group A. The auto‐antibody to LSP was positive in both groups 3 months after the final immunization. Although it was significantly reduced in Group A compared with the level 3 days after the final immunization, it remained high in Group B. Liver damage, production of the auto‐antibody to LSP and DTH to LSP were reduced by adoptive transfer of normal murine spleen cells, but the spleen cells of neonatally thymectomized mice showed no such effects. These results suggest that normal spleen cells possess the capability of suppressing auto‐immune hepatitis, and the defect of this capability due to T‐cell dysfunction by neonatal thymectomy contributes to the promotion and persistence of auto‐immune hepatitis.

Sequential analysis of experimental autoimmune thyroiditis induced by neonatal thymectomy in the Buffalo strain rat

We have studied the evolution of thyroiditis induced by neonatal thymectomy in Buffalo strain rats, with particular emphasis on the thyroid lymphocytic infiltrate. The earliest change was increased endothelial Ia expression, and infiltration of the thyroid at 5 weeks by ED1- and ED2-positive macrophages and B and T cells. The T cells comprised equal numbers of Ox 8 (T cytotoxic/suppressor)- and W3/25 (T helper)-positive cells. Ia-positive thyroid follicular cells were seen only in the presence of a T-cell infiltrate. Thyroglobulin antibody levels, thyroid weight, thyroid follicular cell Ia expression and lymphocytic infiltration of the thyroid were maximal between Weeks 12 and 24 and impairment of macrophage function by injection of silica at this time produced amelioration of disease. The thyroid weight returned to control levels by Week 34 and Ia expression by thyroid cells disappeared. Circulating Ox 8-positive T cells were reduced between Weeks 12 and 24 and by Week 34 had returned to control levels. Our results indicate that the mononuclear infiltrate precedes thyroid follicular cell Ia expression and macrophages play an important role in perpetuating thyroiditis. Recovery from disease is accompanied by a return to normal in circulating suppressor/cytotoxic T cells.

Effect of immunocompetence on the establishment and maintenance of latency with Marek's disease herpesvirus.

Marek's disease virus (MDV) infections normally have an early cytolytic phase in lymphoid organs at 3 to 6 days post-infection followed by a period of latent infection. Little is known about the mechanisms that govern latency with herpesvirus infections, including Marek's disease (MD). To investigate the importance of immunocompetence for either the establishment or the maintenance of latency in MD, immunosuppressive treatments were applied prior to infection with MDV or after latency was established. These included cyclosporin (Cs) or betamethasone (BM) treatments, neonatal thymectomy plus cyclophosphamide treatment (Tx/Cy), and infection at a young age before full competence. The effect of all the treatments was determined by examining tissues and spleen cells for evidence of virus replication before and after cultivation in vitro. Induced (Cs or Tx/Cy treatments) or natural (infection at a young age) incompetence resulted in prolonged and more widespread early cytolytic infection. Immunosuppression by Cs after latency had developed resulted in a reappearance of cytolytic infection in the spleen and it enhanced the cytolytic infection in the thymus and the bursa of Fabricius. After immunosuppression with Cs, cytolytic infection was found mostly in T cells, although many of the virus-positive cells did not have markers for either T cells or B cells. Immunosuppression by BM after latency had developed also resulted in the reappearance of cytolytic infection in the spleen but only at a very low level. These results suggest that immunocompetence is required for the establishment and maintenance of latency.

Immunologic and clinical studies on murine experimental autoimmune gastritis induced by neonatal thymectomy

Experimental autoimmune gastritis (AIG), defined by the appearance of autoantibodies to parietal cells, was induced by neonatal thymectomy in BALB/c nu/+ mice 3 days after birth. Vitamin B12 absorption and intrinsic factor in the stomach extract decreased compared with those in AIG-negative control groups. No decrease of the serum A/G ratio in AIG-bearing mice was observed. Although development of anemia, as evaluated by a decrease in hematocrit value, was poor until 12 mo of age and the gastric mucosa was hypertrophie, the AIG resembled human pernicious anemia rather than Ménétrier's disease. Adoptive transfer of spleen cells, but not sera, of AIG-bearing nu/+ into BALB/c nu/nu mice caused AIG in all animals 1 mo later, indicating the involvement of lymphocytes in the induction mechanism of AIG. Cytofluorometric and immunohistochemical analysis of lymphocytes in the gastric mucosa revealed T-cell infiltration at an early stage (1.5–3 mo) followed by B cell infiltration (6 mo). When the fraction enriched with parietal cells, which were intensively stained with sera of AIG-bearing mice and fluorescent antibody to mouse immunoglobulin G, was injected into the foot pads of AIG-bearing nude mice, typical delayed-type hypersensitivity reaction was observed in all animals. This was not seen in the mice injected with the cell fraction enriched with chief cells, although a few of them were stained by the immunofluorescent technique. Thus, the delayed-type hypersensitivity reaction seems to be directly involved in the mechanism of tissue damage.

Analysis of experimental autoimmune oophoritis as a model of premature ovarian failure

Neonatal thymectomy in mice at 3 days of age (Tx-3) can induce autoimmune oophoritis and results in complete loss of oocyte at young adult age. We examined endocrinological and immunological abnormalities in Tx-3 (C3H/He X A/J)F1 female mice to find some similarities to premature ovarian failure (POF) in humans. The majority of the Tx-3 mice showed irregular estrous cycles during 7 to 9 weeks of age then fell into continuous diestrous. Endocrinological analysis of Tx and sham-Tx mice revealed that serum gonadotropin levels (LH and FSH) of Tx-3 mice rapidly increased from 8 weeks of age and serum estradiol levels significantly decreased from 10 weeks of age (P less than 0.05). In contrast with estradiol, serum androstenedione levels significantly increased from 10 weeks of age (P less than 0.01). In the mice with oophoritis, circulating autoantibodies against ooplasm and/or zona pellucida determined by immunoperoxidase method could be detected from 6 weeks of age, became high titer from one or two weeks later, but resulted in low titer or negative test from about 4 months of age. Acute loss of oocyte with massive mononuclear cell infiltration coincident with the appearance of these autoantibodies were progressed, and then atrophic ovaries with complete destruction of follicles were seen at 3 months of age. This experimental model of autoimmune oophoritis abounds in suggestion for the understanding of one of the possible etiology of POF in women.

Cyclosporine in insulin-dependent diabetes mellitus

It had been known for several years that IDDM (type 1) can be prevented in BB rats or NOD mice by various immunologic interventions, including antilymphocyte serum, l neonatal thymectomy, 2.3 total lymphoid irradiation, 4 or lymphocyte transfusions. 5 However, none of these methods can easily be applied to humans. It was with great interest that it was learned that cyclosporine had the same preventive effect, 6-8 because the d/rug had been Used clinically for >5 years in organ ,~tr~insplantation in a relatively safe fashion. It seemed logical to evaluate the effect of cyclosporine on recent-onset human IDDM. Two pilot studies were performed in Canada and France that led to placebo-controlled randomized trials.

Mechanisms of T cell hyperreactivity in obese strain (OS) chickens with spontaneous autoimmune thyroiditis: lack in nonspecific suppression is due to a primary adherent cell defect.

The disturbed homeostasis of the immune system in Obese strain (OS) chickens with spontaneous autoimmune thyroiditis consists in a general T cell hyperreactivity (concanavalin A hyperresponsiveness, interleukin 2 (IL 2) hypersecretion), particularly expressed by those lymphocytes which infiltrate the diseased thyroid gland. This abnormality has been attributed to a defective regulation of both IL 2 production and IL 2 function by low m.w. factors, which are present in serum and splenocyte culture supernatants of normal chickens, but deficient in the OS. In the present study we identified the cellular origin of IL 2 antagonistic activity as a nonlymphoid, adherent cell. Suppressor factor production in vitro was confined to the plastic adherent fraction of spleen cells and preincubation of splenocytes with nylon wool, silica particles, or carbonyl iron significantly reduced the nonspecific suppressive activity of the culture supernatant. Kinetic studies revealed the defect in nonspecific suppression to entail prolonged IL 2 production by concanavalin A-activated OS spleen cells. In vivo treatment of normal White Leghorn chickens with silica led to a decrease in suppressive serum activity down to the OS level, whereas neither neonatal thymectomy nor bursectomy had any effect. The defective suppressor factor production in autoimmune chickens appeared to be due to a functional, but not numeric defect of macrophages as revealed by phenol red staining. The possibility that this aberration in adherent cell function might be a secondary phenomenon to the recently described reduced corticosterone tonus in OS chickens was excluded by in vivo substitution with exogenous glucocorticoids which did not normalize the suppressor defect in serum or in conditioned medium. Finally, we present evidence that T lymphoblasts from OS animals are less susceptible to IL 2 antagonistic regulation than normal cells, which possibly further contributes to the T cell hyperfunction of this autoimmune strain.

Defective differentiation of natural killer cells in SJL mice. Role of the thymus.

As previously shown, three distinct phenotypes exist in murine natural killer (NK) cell activity when it is evaluated by the endogenous levels of activity and the susceptibility to augmentation by interferon (IFN) and IFN inducers. The "low" phenotype has low levels of activity which can be poorly augmented by IFN, as in mice of SJL strain. The "inducible" phenotype exhibits low endogenous levels but can vigorously respond to IFN-mediated augmentation, as in A.SW strain. The "high" NK phenotype shows high levels of endogenous activity which can be augmented to still higher levels by IFN, as in B10.S mice. Since SJL mice with congenital absence of the thymus (nude) were of the inducible type, the effect of neonatal thymectomy was examined in the present study. Neonatal thymectomy was found to convert the low phenotype of SJL mice to the inducible, mimicking the effect of nu/nu genotype. Thymectomy as late as 25 days after birth was effective, but retransplantation of a syngeneic newborn or adult thymus, or thymocytes, failed to reverse the effect of thymectomy. The poor responsiveness of NK activity to IFN in SJL, therefore, is extrinsic to the NK cell lineage and is attributable to suppression or maturational block of NK cell differentiation by the thymus during the first few weeks of neonatal life. A series of experiments with bone marrow chimeras showed that the SJL recipients did not allow the expression of inducible or high phenotype by bone marrow progenitors from allogeneic donors with either phenotype. Therefore, the SJL recipients provide an environment which suppresses not only the development of IFN-sensitive NK cell precursors, but also the levels of endogenous NK cell activity. SJL bone marrow cells gave rise to NK activity of inducible phenotype in B10.S recipients, confirming the crucial role of the environment in which NK cell differentiation takes place.

Immunotherapy for virus infection.

Understanding of virus-induced immune response disease and the related immunopathology has its roots in the studies reported by Rowe (1954). He showed that suppression of immune responses changed the ordinarily lethal, acute infection with lymphocytic choriomeningitis virus (LCMV) to a persistent infection in susceptible mice. The subsequent work of many investigators who used neonatal thymectomy, genetically athymic mice, irradiation, antilymphoid drugs, or antithymocyte sera, etc. (reviewed in Buchmeeer et al. 1980) extended the concept of immune response-mediated injury during viral infection. Thereafter, the role of lymphocytes in mediating virus-induced immunologic injury was delineated. First, experiments performed independently by Lundstedt (1969) and Oldstone et al. (1969) showed that lymphocytes obtained 6–9 days after an acute LCMV infection or primary inoculation killed LCMV-infected targets in vitro. Next, such lymphocytes were found to be of thymic origin and to bear Thy 1.2 markers of their surfaces (Cole et al. 1973; Marker and Volkert 1973). Gilden et al. (1972a, b) showed that mice which survived an ordinarily lethal dose of LCMV owing to immunosuppression developed acute lymphocytic choriomeningitis (LCM) disease and died when reconstituted with syngeneic, immune T-lymphocytes. Zinkernagel and Doherty (1974 a) defined the need for two signals between the cytolytic T-lymphocyte and its infected target, namely, virus specificity and H-2 restriction, so that killing of LCMV-infected cells could proceed.

Role of T-Cell Subsets in the Destruction of Established Metastases of 13762A Rat Mammary Adenocarcinoma<xref ref-type="fn" rid="FN1">2</xref>

The 13762A rat mammary adenocarcinoma metastasizes with high frequency to regional lymph nodes and lungs. The intratumoral injection of Corynebacterium parvum on day 7 followed by primary tumor excision on day 20 significantly prolonged survival and cured 10-40% of syngeneic F344 rats. Established metastases were destroyed by the treatment, and strong and specific tumor rejection immunity was induced. The purpose of the present study was to determine if T-cells were required for the C. parvum treatment to be effective and to identify the subsets of T-lymphocytes that might participate in the response. The results indicated that rats depleted by either neonatal thymectomy or a combination of adult thymectomy, 900 rad, and bone marrow reconstitution did not inhibit tumor growth after C. parvum treatment. Restoration of depleted rats with lymph node cells permitted effective treatment. The lymph node cells that were responsible for restoration expressed both W3/13 (pan-T-cell) and W3/25 (helper T-cell) membrane-associated differentiation antigens. T-cells that bore the MRC OX8 (cytotoxic-suppressor T-cell) antigen did not restore the response to C. parvum treatment. The effect of lymph node restoration was markedly potentiated by simultaneous administration of thymocytes, a T-cell population that expresses both W3/25 and MRC OX8 antigens. In conclusion, the cytotoxic-suppressor T-cells were ineffective in the restoration of T-cell-depleted, tumor-bearing rats to benefit from C. parvum but helper T-cells were highly effective, and their activity was strongly potentiated by administration of thymocyte amplifier cells.

Human tumour xenografts growing in immunodeficient mice: a useful model for assessing chemotherapeutic agents in bronchial carcinoma.

Xenografts from eight human bronchial carcinomas have been established in CBA/Lac mice rendered immunodeficient by neonatal thymectomy followed three weeks later by whole body irradiation (7.35 Gy (735 rads)) after a priming dose of cytosine arabinoside. Growth rates of individual tumour lines remain constant and the histological and chromosomal characteristics of the original tumour are maintained through multiple serial passages over many months. With specific growth delay as the principal end point this system may be used to assess the response of histologically different lung tumours to chemotherapeutic agents.

Hypertrophic gastritis with hypergastrinemia and protein loss after neonatal thymectomy in mice.

Hypertrophic gastritis, histologically characterized by a depletion of parietal and chief cells and by varying degrees of lymphocyte infiltration along the thickened muscularis mucosa, could be induced by neonatal thymectomy (Tx) without any additional treatment in about 50% of mice (C3H/HeMs X 129/J)F1 (C3.129). The thickness of the mucosa in gastritic mice increased with age, forming giant folds. In Tx mice with an early stage of abnormal mucosal folds at 6 months of age, numbers of parietal cells per mucosal tissue unit area (parietal cell densities) and ratios of parietal cells to mucous cells became lower than in control mice, and serum gastrin levels became contrastingly higher with the increasing severity of gastritis. Circulating antibodies against parietal cells (APA) were detected by indirect immunofluorescence (IFL) in the mice. A good correlation was observed between APA and gastritis: APA with high titers (more than 1,000-fold dilutions) appeared when severe lesions were found. In mice with giant mucosal folds at 18 months of age, serum protein levels were within normal limits, but fecal clearance rates of 125I-labelled polyvinylpyrrolidone (125I-PVP) were significantly increased. These results suggest that the hypertrophic gastritis induced by neonatal Tx is characterized by hypergastrinemia due to parietal cell depletion caused by the presence of circulating APA and the protein loss from the hypertrophic mucosa. Both histological and physiopathological similarities were found between the gastritis in the mice and Menetrier's disease in man.

Pathology of the thymus in immunodeficiency states.

The discovery of significant alterations of the thymus at autopsy of children deceased from repeated infections (GITLIN and CRAIG 1963; NEZELOF 1968; TOBLER and COTTIER 1958) was followed within 2 or 3 years by the publication of experimental data obtained from neonatal thymectomy in mice and largely contributed to establishing in humans the key role of the thymus in the development of the lymphoid tissue and cellular immunity (for review see MILLER 1979).

Effect of antilymphocyte serum and neonatal thymectomy upon onset of diabetes in the Chinese hamster.

Prediabetic Chinese hamsters were treated with antilymphocyte serum (ALS), or thymectomized in order to test the hypothesis that beta-cell loss leading to diabetes in this animal model was related to cell-mediated autoimmunity. In addition, passive transfer of diabetes from the Chinese hamster to the nude mouse was attempted by transplantation of lymphocytes. Treatment of prediabetic Chinese hamsters with ALS or thymectomy did not alter development or severity of diabetes in this animal model. Lymphocytes from newly diagnosed diabetic Chinese hamsters did not cause hyperglycemia in nude mice. These three lines of evidence suggest that cell-mediated autoimmunity does not contribute to the etiology of diabetes in the Chinese hamster. The Chinese hamster remains a good model for the study of those forms of diabetes not related to cell-mediated autoimmune phenomena.

Contact sensitivity to DNCB in normal and cell-mediated-immunity deficient chickens: In vivo detection and correlation with lymphocyte transformation and graft-versus-host reaction

Optimum conditions for the contact sensitivity (CS) test using 2,4-dinitrochlorobenzene (DNCB) were sensitization with 0.25 ml of a DNCB solution (10 mg/ml) and challenge with one-tenth of this dose two weeks later. This produced reaction in terms of skin thickness which could be easily observed and measured. The cutaneous hypersensitivity developed slowly, reaching its maximum manifestation after 24 h of challenge and gradually declining thereafter. The development of a skin reaction was typical of delayed type hypersensitivity and was characterized histopathologically by congestion, oedema, mononuclear and heterophilic cell infiltration in the dermal layer and lymphocytic perivascular cuffing. The efficiency of the CS test was compared with that of the lymphocyte transformation (LT) test and graft-versus-host (GvH) reaction for monitoring cell-mediated immunity (CMI) in normal and CMI deficient chickens. CMI deficient chickens were prepared by neonatal thymectomy and inoculation of antithymocyte serum. The CMI response of deficient chickens was significantly less ( p < 0.01) as determined by all three tests. The percentage decrease in response to mount GvH, CS and LT was 88.9, 58.3 and 74.6, respectively. All the tests were found to be equally effective in assessing CMI response in chickens as determined by comparing the means of their performances.

The role of cytostasis in antitumor immunity: Comparison between syngeneic and allogeneic systems

Different aspects of the immune response differ with respect to sensitivity of neonatal thymectomy (NTx). The effect of thymectomy on Days 1 and 7 after birth (NTx-1, NTx-7) on antitumor immune responses to both a syngeneic and an allogeneic tumor graft was examined. The tumor used was the same, Meth A fibrosarcoma of BALB/c origin, but the mice inoculated differed. The results show that the immune response to a syngeneic tumor graft is primarily cytostatic rather than cytolytic and is relatively insensitive to neonatal thymectomy, while the response to a large dose of allogeneic tumor cells includes a cytolytic mechanism of high thymus dependence.

Adoptive transfer of autoimmune diabetes mellitus in biobreeding/Worcester (BB/W) inbred and hybrid rats.

Adoptive transfer of diabetes was accomplished by the injection of Con A-activated acutely diabetic BB/W rat spleen cells into immunosuppressed diabetes-resistant BB/W control rats and F1 hybrid offspring produced by BB/W X Lewis, BN, Yashida, and NEDH matings. Immune suppression methods that facilitated adoptive transfer of diabetes included neonatal thymectomy, cyclophosphamide, and splenectomy plus rabbit anti-rat lymphocyte serum injections. The successful transfer of BB/W diabetes to otherwise normal (BB/W X inbred)F1 rats and to diabetes-resistant BB/W animals suggests that antigenically normal pancreatic beta cells were destroyed by the injected effector cells. Diabetes-resistant BB/W control rats also evidenced diabetes after the injection of cyclophosphamide alone. The requirement for immunosuppression suggests that an intact immune system protects against adoptive transfer and diabetes in diabetes-resistant BB/W rats.

Studies on the role of the thymus in the selection and clonal expansion of T-cell precursors.

Since the observation that neonatal thymectomy in mice causes a severe and definitive impairment of cell mediated immune function1 there has been little doubt that the thymus plays a central role in the development of the fully competent T-lymphocyte system. While events such as the expression of antigen specific receptors2, the acquisition of MHC restriction and self tolerance3 as well as the emergence of phenotypically divergent functional subpopulations4 are all accepted as features of T-cell ontogeny our understanding of the influence of the thymus upon, and its mechanistic involvement in the differentiation process has progressed little. However it remains attractive to view the thymus as providing the primary site for the selection and amplification of those differentiating T-cell clones physiologically capable of functioning with the self environment of the host. Such a view supposes a series of interactions between the thymic epithelial matrix and the colonizing lymphohaematopoietic cells which include the T progenitors, macrophages and a population of dendritic cells5–7. Within this thymic milieu cell surface MHC gene products may act as ligands to provide signals for the selection of thymocytes of relevant specificity while soluble factors such as interleukins may induce precursor proliferation.

Ovarian dysfunction in neonatally thymectomized rats.

The effect of neonatal thymectomy upon the reproductive function was examined infemale Wistar rats. After female pups were thymectomized within 48 hours of birth, the somatic growth was not inferior to that of controls after 18 days of age, but less than controls from 3 to 15 days of age (P<0.01 or 0.05). The day of vaginal opening in thymectomized rats (41.2±1.02) was similar to that of intact rats (39.9±0.78). The results of incidences of vaginal estrous and proestrous stageor estrous stage alone at 20-day in-tervals from 50 to 169 days of age in thymectomized rats indicated that the ovarian func-tion appeared to decline progressively after 90 days of age. Between thymectomized and intact rats, there was no significant difference in the weight of ovaries, uteri, pituitary gland, thyroid glands and spleen, except the weight of adrenal glands (P> 0.05) at 180 days of age. Histological findings in thymectomized rats indicated the hyperplasia of theinter-stitial cell elements and degeneration of follicles in the ovary and formation of marked cytoplasmic haloes in the basophilic cells of the pituitary gland.

Studies of lymphoproliferation in MRL-lpr/lpr mice.

MRL-lpr/lpr mice develop massive lymphoproliferation and an associated autoimmune process that includes anti-DNA formation, vasculitis, and glomerulonephritis. We have investigated the lymphoproliferation of MRL-lpr/lpr mice and have found that multiple factors are operative. Although neonatal thymectomy markedly retards the syndrome, chronic injection of poly rI.rC could substitute for the thymus. The resulting cells had the phenotype characteristic of the abnormal MRL-lpr/lpr T cells, Thy-1+, dull Ly-1+, Lyt-2-, 6B2+, Ig-. Splenectomy at 2 wk of age markedly retarded the development of this syndrome; however, splenectomy at birth did not. Some protection was afforded by splenectomy at 5 wk. Thus, there appears to be a critical period in the life of an MRL-lpr/lpr mouse when the spleen contributes importantly to the lymphoproliferation. A most remarkable observation was that an MRL-lpr/lpr spleen graft under the kidney capsule could induce lymphadenopathy characteristic of lpr/lpr mice in MRL +/+ recipients. Cells within the graft had to be able to proliferate for the adenopathy to occur because irradiation of the spleen with 800 R just before grafting abrogated the lymphadenopathy-inducing potential. No adenopathy was induced by +/+ spleen grafts placed into +/+ mice. Although MRL-lpr/lpr males develop disease slightly more slowly than female littermates, the differences are small. Manipulations that retard disease, such as splenectomy at 2 wk or neonatal thymectomy, magnified the sex differences. Male MRL-lpr/lpr mice that were thymectomized and splenectomized and given polyclonal immune activators failed to develop either anti-DNA or lymphadenopathy, whereas their female littermates expressed both abnormalities. We conclude from these studies that multiple factors serve to modulate the magnitude of the lymphoproliferation and autoimmune syndrome of MRL-lpr/lpr mice.