Quantitative studies of lymphocytes and other cell populations in the bone marrow of neonatally thymectomized C3H mice

The effects of neonatal thymectomy on the development of the lymphoid, erythroid and granulocytic cell populations in mouse bone marrow have been assessed by quantitative techniques. The numbers per unit volume of bone marrow of 17 cell types were determined in neonatally thymectomized and sham thymectomized C3H mice at two, four and eight weeks of age, and compared with those of normal C3H mice. After neonatal thymectomy the numbers of small lymphocytes, large and medium-sized lymphoid cells, and erythroid cells reached normal levels at two weeks but fell progressively to 18%, 22% and 42% of normal, respectively, by eight weeks. In sham thymectomized mice these cell populations did not differ significantly from normal. Immature and mature granulocytes were elevated in numbers two weeks after either neonatal thymectomy or sham thymectomy, suggesting a transient non-specific stimulation of granulocytopoiesis. During continuous infusion of ³H-thymidine for ten days in neonatally thymectomized mice aged four weeks and eight weeks many bone marrow small lymphocytes remained unlabeled. The results demonstrate that early postnatal development of bone marrow lymphoid and erythroid cells proceeds normally in the absence of the thymus, in accord with the concept of the bone marrow as a primary site of lymphocyte production and differentiation. In addition, some slowly-renewing small lymphocytes in bone marrow appear to be thymus-independent cells.     

The immunological responsiveness of germ-free mice thymectomized at birth. II. Lymphoid tissue and histopathology

The effect of neonatal thymectomy and antigenic stimulation on the lymphoid cell population has been studied in germ-free mice. Neither thymectomy nor injection of sheep erythrocytes induced any significant alteration in the blood lymphocyte levels. There was a clear-cut reduction in the cellularity of the periarteriolar lymphocyte sheaths of the spleen and of the paracortical regions of the lymph nodes in the thymectomized mice. Following stimulation with sheep erythrocytes, large pyroninophilic cells appeared in these areas in the intact germ-free controls but in only a few thymectomized mice and then in reduced numbers. Thymectomy did not influence the cellularity of the lymphoid follicles but less germinal centre and plasma cell activity occurred in response to an injection of sheep erythrocytes. Lesions suggestive of autoimmune reactivity were not found in lymphoid or nonlymphoid tissues of neonatally thymectomized germ-free mice. Lesions typical of viral infections were seen in some germ-free mice in both thymectomized and intact groups. It is concluded that the specific defect associated with the absence of the thymus is a reduction in a particular class of lymphocytes the development of which is under thymus control and the activities of which are to mediate certain defined immunological responses.     

Changes produced by pertussis antigen on the blood cells and lympho-haemapoietic tissues after early and late thymectomy or splenectomy

The effect of thymectomy and splenectomy in C3H/Bi mice on the responses of circulating leucocytes and on morphological changes of the haematopoietic tissues after injection of pertussis vaccine has been studied.

After pertussis all mice showed depletion of lymphoid cells in all the lymphoid organs as well as in bone-marrow and an increased number of leucocytes, lymphocytes, neutrophils and monocytes in the circulation. Neonatal thymectomy decreased lymphocytosis produced by pertussis. Thymectomy, at all ages studied, fostered an increase in the number of monocytes and polymorphonuclears in circulation. Splenectomy at birth or early in life provoked an increase in levels of circulating polymorphonuclears and lymphocytes in pertussis treated animals.

In neonatally thymectomized mice the depletion of lymphoid cells from lymphoid tissues after pertussis could be shown to include the thymic-independent areas. The depletion of small lymphocytes from thymus following pertussis persisted longer than depletion of small lymphocytes from spleen, marrow or lymph nodes. The longer persistence of lymphoid depletion in the thymus than in peripheral lymphoid tissues is, we believe, to be related to the central lymphoid function of thymus as a site of differentiation of lymphoid cells and to the aloofness of thymus from recirculation of fully differentiated peripheral lymphocytes.

     

Size of the population of CD4+ natural killer T cells in the liver is maintained without supply by the thymus during adult life

Given that there are few natural killer T (NKT) cells in the liver of athymic nude mice and in neonatally thymectomized mice, it is still controversial whether all NKT cells existing in the liver are supplied by the thymus or if some such cells develop in the liver. To determine whether or not NKT cells are consistently supplied from the thymus during adult life, thymectomy was conducted in mice at the age of 8 weeks. Interestingly, the proportion and number of CD4+ NKT cells increased or remained unchanged in the liver after adult thymectomy and this phenomenon continued for up to 6 months after thymectomy. The administration of alpha-galactosylceramide induced severe cytopenia (due to apoptosis) of CD4+ NKT cells in the liver on day 1, but subsequent expansion of these NKT cells occurred in thymectomized mice similar to the case in normal mice. However, in thymectomized mice given lethal irradiation (9.5 Gy) and subsequent bone marrow transfer, the population of CD4+ NKT cells no longer expanded in the liver, although that of CD8+ NKT cells did. These results suggest that thymic CD4+ NKT cells, or their progenitors, may migrate to the liver at a neonatal stage but are not supplied from the thymus in the adult stage under usual conditions. CD8+ NKT cells can be generated in the liver.     

Development of Natural Killer Cell Activity and Genetic Resistance to Bone Marrow Transplantation with Age: Effect of Neonatal Thymectomy

The effect of neonatal thymectomy on the development of splenic and bone marrow natural cell-mediated cytotoxicity and on genetic resistance to bone marrow transplantation was examined in mice. Natural cytotoxicity was measured by a ⁵¹Cr release assay; the ability to engraft foreign bone marrow was assayed by the spleen colony method. The natural cytolytic response of spleen cells increased progressively from youth to early adulthood, whereas that of the bone marrow declined during the same age period. Neonatal thymectomy significantly elevated the natural killer cell response of young mice only (4 weeks, spleen; 6 weeks, bone marrow). In other experiments, neonatally thymectomized and sham-operated mice were lethally irradiated at 4 or 6 weeks of age and injected with 2.5, 5.0 or 10 million rat marrow cells. Six days later spleen colonies were markedly reduced in both 4- and 6-week-old neonatally thymectomized mice with all rat marrow cell doses tested. Neonatal thymectomy did not alter the percentage of erythroid versus other colonies at either 4 or 6 weeks. In both thymectomized and sham-operated mice the number of colonies increased with increases in marrow cell dose. The data are suggestive of a production and dissemination to the spleen of cells involved in the natural cytotoxic response from the bone marrow.     

The role of the thymus in development of lympho-hemopoietic tissues. The effect of thymectomy on development of blood cells,bone-marrow,spleen, and lymph nodes

The effect of thymectomy at different ages in C3H mice on development of circulating leukocytes and cells of marrow, spleen and lymph nodes has been analyzed. Regardless of the age at which thymectomy is performed depression of numbers of circulating lymphocytes is produced. Thymectomy at birth did not affect significantly the relative number of lymphocytes in the marrow during the first few weeks of life, later they fell to low levels. Thymectomy at four weeks was followed by prompt reduction in relative numbers of lymphocytes in the marrow. After reaching six weeks of age, neonatally thymectomized mice showed a high proportion of monocytes in the marrow. Neonatal thymectomy and thymectomy at two weeks of age reduced the number of eosinophils in the marrow. Neonatal thymectomy inhibited development of lymphocytes in the spleen, whereas thymectomy later in life produced only transient depression of lymphocytes in this organ. In addition, neontal thymectomy decreased the relative numbers of small lymphocytes in the lymph nodes. This was associated with drastic depletion of lymphocytes in the deep cortical regions of the nodes.     

Changes in human T lymphocytes after thymectomy and during senescence

Peripheral lymphocytes from individuals who had been thymectomized in adult life for myasthenia gravis (MG) or for other, nonimmunological reasons showed a moderate decrease in proliferative response capacity to several T-cell mitogens as compared to lymphocytes from normal individuals. The decrease of the response to mitogens and allogeneic lymphocytes was 20–30% within 5 years after thymectomy and about 50% more than 15 years after thymectomy. A comparable decrease in lymphocyte proliferative response capacity was found in healthy aged humans (68–97 years old). Analysis of T lymphocytes from both aged and thymectomized individuals with monoclonal (OKT) antibodies showed a similar pattern: the proportion of T lymphocytes binding OKT3 was reduced, and the OKT4/OKT8 ratio was increased. Hardly any T lymphocytes binding OKT6, OKT10, or OKT1 were found. A biochemical parameter for human T-cell differentiation, the lactate dehydrogenase (LDH) isoenzyme pattern, showed a significantly lower H/M ratio in the group of elderly people compared to young individuals. Furthermore, among patients thymectomized for MG, a significant correlation was observed between the LDH isoenzyme pattern of the T lymphocytes and the proliferative response to mitogens of these cells. In contrast, in healthy thymectomized individuals the LDH isoenzyme pattern appeared to be normal. These findings indicate that, after thymectomy or involution of the thymus, at least part of the peripheral blood T lymphocytes have properties different from those of the cells of young individuals. These cells might represent immature and/or not fully differentiated lymphocytes.     

Theta-bearing cells in the bone marrow of thymus-deprived mice. Numbers and nature.

Theta-bearing cells in lymphomyeloid tissues of thymus-deprived and normal mice have been studied by the use of anti-theta antiserum and cytotoxicity tests in addition to functional tests. In contrast to the findings in peripheral lymphoid tissues, increased percentages and numbers of theta-bearing cells were found in the bone marrow of neonatally and nude mice as compared with normal and sham-thymectomized mice. In adult thymectomized mice, percentages comparable to those in sham-operated littermates were found. The findings were not due to irrelevant antibodies in the anti-theta antiserum, and neonatally thymectomized mice grafted with a thymic lobe showed percentages of theta-positive cells in the bone marrow comparable to those of sham-operated animals. Adrenalectomy did not lead to diminished percentages of theta-positive cells in the bone marrow of neonatally thymectomized mice, and the serum levels of hydrocortisone and corticosterone were within normal ranges in thymus-deprived mice. The mitogen responses and graft-versus-host activity of bone marrow cells from neonatally thymectomized mice suggest that most theta-positive cells in the bone marrow of these mice are functionally immature cells.     

Theta-Bearing Cells in the Bone Marrow of Thymus-Deprived Mice

Theta-bearing cells in lymphomyeloid tissues of thymus-deprived and normal mice have been studied by the use of anti-theta. antisenun and cytotoxicity tests in addition to functional tests, In contrast to the findings in peripheral lymphoid tissues, increased percentages and numbers of theta-bearing cells were found in the bone marrow of neonatally thymectomized and nude mice as compared with normal and sham-thymectomized mice. In adult thymectomized mice, percentages comparable to those in sham-perated littermates were found. The findings were not due to irrelevant antibodies in the anti-theta antiserum, and neonatally thymectomized mice grafted with a thymic lobe showed percentages of theta-positive cells in the bone marrow comparable to those of sham-operated animals. Adrenalectomy did not lead to diminished percentages of theta-positive cells in the bone marrow of neonatally thymectomized mice, and the serum levels of hydrocortisone and corticosterone were within normal ranges in thymus-deprived mice. The mitogen responses and graft-versus-host activity of bone marrow cells from neonatally thymectomized mice suggest that most theta-positive cells in the bone marrow of these mice are functionally immature cells.     

Acceleration of the onset of diabetes in NOD mice by thymectomy at weaning

The effect of thymectomy performed at weaning (3 weeks) and at 6-7 weeks of age on the incidence of diabetes was examined in the non-Obese diabetic (NOD) mouse, a spontaneous model of immunologically mediated insulin-dependent diabetes similar to human type I diabetes. When performed at weaning, thymectomy led to a dramatic increase in the incidence of diabetes in NOD females in comparison to sham-thymectomized animals. Conversely, no change in the incidence of the disease or the expression of insulitis was noted when thymectomy was performed in NOD males. When delayed beyond 6-7 weeks of age, thymectomy had no effect on NOD males and females. Flow cytometry analysis of spleen cells from intact mice and mice thymectomized at weaning or at 6-7 weeks of age demonstrated a significant depletion of the T cell subsets in both groups of thymectomized animals. These results indicate that the onset of diabetes in NOD mice is submitted to thymic regulation and that the T cell depletion induced by thymectomy at weaning accelerates the disease, an effect possibly due to the loss of some T cell-dependent suppressor mechanisms.     

Consequences of neonatal thymectomy in New Zealand black mice

The possible role of the thymus in autoimmune disease was studied by comparing the effects of neonatal thymectomy on New Zealand Black (NZB) mice (which develop a Coombs positive haemolytic anaemia) and on C3H/Bi, F₁ (C57BL × C3H/Bi), C57BL and TO mice.

The neonatally thymectomized NZB mice, in common with those of other strains, showed lethal wasting, a lymphocyte deficit in their lymphoid organs and blood, their packed cell volume was reduced and some had liver lesions associated with the hepatotrophic virus MHV-1. As in C3H/Bi and hybrid mice, thymectomy had little effect on the levels of immunoglobulins (IgG, IgA, IgM) present in their sera.

Removing the thymus from NZB mice did not prevent and could precipitate Coombs positive reactions; thymectomized mice of the other strains were Coombs negative. Although germinal centres develop and plasma cells occur in the lymphoid organs of most thymectomized mice, the striking feature of the NZB mice was the large number, size and activity of the germinal centres that persisted after thymectomy.

     

Post-thymectomy Murine Experimental Autoimmune Oophoritis Is Associated With Reduced Natural Killer Cell Activity

PROBLEM: Natural killer (NK) cells can influence the immune response by secreting potent lymphokines. It has been suggested that NK cells have a suppressive action on B cells, and that impaired NK cell activity may play a role in some types of autoimmunity. NK cell abnormalities have been reported in women with premature ovarian failure. We therefore examined NK cell activity during the development of murine experimental autoimmune oophoritis, which serves as a model for autoimmune ovarian failure in women. METHOD OF STUDY: Neonatally thymectomized and sham-operated C57B1/6 × A/J (B6A) mice were prepared and sacrificed at 4, 6, 8, and 10 weeks after surgery. Splenic NK cell activity was determined in groups of five or more mice by measuring the percent specific lysis of target YAC-1 lymphoma cells using a standard 4-hr chromium release cytotoxicity assay. The number of splenic NK cells in neonatally thymectomized and sham-operated animals was also compared using flow cytometry. In a subsequent experiment, interleukin 12 (IL-12; NK cell-stimulating factor) was administered to neonatal mice before neonatal thymectomy. RESULTS: Neonatally thymectomized mice with associated autoimmune oophoritis had a 75% reduction in the number of splenic NK cells, and 50% or greater reduction in splenic NK cell activity at 4, 6, and 8 weeks after surgery. IL-12 treatment before neonatal thymectomy maintained NK cell activity and was shown to ameliorate the associated autoimmune oophoritis. CONCLUSION: Murine post-thymectomy autoimmune oophoritis is associated with reduced NK cell number and impaired NK cell activity, and in these respects the model is similar to premature ovarian failure in women. Research to define the relationship between NK cell abnormalities and the mechanism of ovarian failure in this model might lend insight into the pathogenesis of premature ovarian failure in women.     

Immunopathology of New Zealand black mice treated with antilymphocyte globulin

Antilymphocytic globulin (ALG) suppressed the development of haemolytic anaemia with positive antiglobulin (Coombs) reactions in intact and adult thymectomized New Zealand Black (NZB) mice, although some recipients eventually became positive after treatment was stopped. Macroglobulinaemia and lymphoid cell infiltrates were resistant and complex nephritis was unaffected by, and in thymectomized recipients accelerated by, ALG, whilst many such recipients developed severe Coombs negative haemolytic anaemia. Splenomegaly was less pronounced in ALG treated than in control NZB mice. However, lymphoproliferative changes in general, and reticulum cell hyperplasia in particular, progressed despite ALG treatment in early life. On the other hand, this agent did not provoke overt neoplasia in the lymphatic or other tissues. Syngeneic lymphoid cells from young donors did not influence the disease of ALG treated mice but rat lymphoid cells in thymectomized recipients induced a temporary suppression of macroglobulinaemia and reticulum cell proliferation was reduced.     

Distribution of immunoglobulin-containing cells in normal and neonatally thymectomized rats

The distribution of immunoglobulin-containing cells (ICC) of the immunoglobulin A (IgA), IgG, and IgM isotypes was examined in various lymphoid and secretory tissues of rats. The effect of neonatal thymectomy of rats on T cells, B cells, and ICC in these tissues was determined by immunofluorescence. The results showed that although T cells were severely depleted in both lymphoid and secretory tissues of the thymectomized (Tx) rats, Tx and normal rats showed comparable numbers of B cells staining for IgA, IgG, and IgM. After neonatal thymectomy, IgA ICC in both lymphoid and secretory tissues were significantly decreased. However, the Tx rats exhibited a compensatory increase in IgM ICC in the identical tissues. Local injection of normal and Tx rats with Streptococcus mutans 6715 resulted in an increase in all isotypes of ICC in the secretory tissues. Although the primary increase in normal rats was due to IgA ICC, Tx rats exhibited the greatest change in the number of IgM ICC.     

Regeneration of thymus grafts. II. Effects on immunological capacity

Mice thymectomized at birth were grafted at 1 week of age with thymus tissue under the kidney capsule. The implants were excised after a period of 1, 2 or 3 weeks and the response of the mice to sheep erythrocytes and to allogeneic skin grafts was tested. Thymectomized mice that had not received thymus implants had twenty times less antibody-plaque-forming cells per million spleen cells than sham-thymectomized controls and failed to reject foreign skin. Some evidence of restoration of immune capacities was obtained in mice bearing for 1 to 2 weeks either normal thymus implants or thymus tissue irradiated in vitro with 500 R. By contrast, thymus tissue irradiated with 2000 R failed to influence neonatally thymectomized mice with respect to their immunological capacities. Most thymectomized mice bearing thymus implants, whether normal, irradiated with 500 or 2000 R, had blood lymphocyte levels within the normal range. Cytological analysis of the lymph nodes and spleen of mice bearing normal thymus implants revealed only very few thymus-derived cells but the proportion of these cells was significantly increased after specific immunization. No thymus-derived cells were however detected in the lymphoid tissues of mice bearing irradiated thymus implants, not even in those that were capable of responding to antigenic stimuli. It is concluded that the thymus plays an essential role in inducing the differentiation of immunologically competent cells from non-competent precursors and that this function is dependent on the integrity of the thymus epithelial-reticular cells.     

Effect of proline-rich polypeptide on experimental autoimmune response to erythrocytes

PRP administration in parallel with RRBC injections increased the AEAP in mice, as did adult thymectomy carried out six weeks before the RRBC injections. On the other hand, PRP administration to thymectomized mice during immunization with RRBC decreased the intensity of AEAP to the level observed in intact, RRBC immunized controls. The number of ARFC among non-B lymphocytes in peripheral blood was increased in mice immunized with RRBC compared to those in non-immunized animals. PRP administration during immunization with RRBC or adult thymectomy lowered their number below the values in non-immune animals and non-B ARFC number comparable to those in control, immunized animals was observed in thymectomized mice injected with PRP in parallel with RRBC. The values of ARFC among the NAL were higher than among the non-B lymphocytes but their shifts in the individual experimental groups were in the same direction as among the non-B lymphocytes. However, their shifts after thymectomy and/or PRP treatment were in opposition to those of the intensity of AEAP in the respective experimental groups.     

Accelerated age-dependent decline in the T suppressor capacity of SJL mice

Tolerance induction with rabbit gamma-globulin was employed as a probe for age-dependent changes in suppressor capacity of SJL lymphoid cells. The tolerant state was assessed by loss of cooperative capacity and by infectious tolerance. The supply of precursor cells was assessed by thymectomy and by treatment with colchicine and cyclophosphamide, which have been reported to eliminate suppressor cells. Thymectomy, 16-18 days before tolerance induction, did not affect antibody response or tolerance inducibility; thymectomy, 33 days before tolerance induction, reduced both antibody response and tolerance inducibility. Colchicine, injected together with aggregate-freed rabbit gamma-globulin, inhibited tolerance induction partially in 35-day-old mice and completely in 106-day-old mice. Colchicine, given to younger mice, thymectomized 17 days before tolerance induction, prevented tolerance induction completely. A low dose of cyclophosphamide interfered with tolerance induction in older, but not in younger mice. A high dose of cyclophosphamide interfered with tolerance induction in thymus cells of younger and older mice. After thymectomy, there was a much more profound interference of a low dose of cyclophosphamide with tolerance induction. Results were discussed in terms of an age-dependent decline of thymus progenitors and of peripheral progenitors of suppressor cells.     

Kinetics of intraepithelial lymphocytes in the small intestine of thymus-deprived mice and antigen-deprived mice.

The kinetics of lymphoid cells within the epithelium of the small gut has been studied in various thymus-deprived mice and in antigen-deprived mice by the use of 3H-thymidine injections and radioautography. In thymus-deprived mice--including adult thymectomized, thymectomized and irradiated, neonatally thymectomized, and nude mice - and in germ-free mice decreased numbers of intraepithelial lymphocytes (IL) were found. On the other hand, the radioautographic results indicated that the remaining IL populations included both newly formed and long-lived lymphoid cells in the same percentages as found in sham-operated controls and normal mice. It is concluded that although the presence of the thymus and the antigen content of the gut is of importance to the maintenance of the numbers of cells in the lymphoid populations of the intestinal wall, the basic kinetics of these cell populations are preserved in deprived mice.     

Kinetics of intraepithelial lymphocytes in the small intestine of thymus-deprived mice and antigen-deprived mice

The kinetics of lymphoid cells within the epithelium of the small gut has been studied in various thymus-deprived mice and in antigen-deprived mice by the use of ³H-thymidine injections and radioautography. In thymusdeprived mice —including adult thymectomized, thymectomized and irradiated, neonatally thymectomized, and nude mice —and in germ-free mice decreased numbers of intraepithelial lymphocytes (IL) were found. On the other hand, the radioautographic results indicated that the remaining IL populations included both newly formed and long-lived lymphoid cells in the same percentages as found in sham-operated controls and normal mice. It is concluded that although the presence of the thymus and the antigen content of the gut is of importance to the maintenance of the numbers of cells in the lymphoid populations of the intestinal wall, the basic kinetics of these cell populations are preserved in deprived mice.