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.     

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.     

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.     

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.     

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.     

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.     

Bone marrow origin of complement-receptor lymphocytes

Substantial regeneration of complement-receptor lymphocytes (CRL) took place in lymph nodes of adult thymectomized whole-body X-irradiated mice reconstituted with semi-allogeneic (F₁) bone marrow. In the presence of complement, alloantisera directed against the transplanted bone marrow killed more than 95% of the lymphoid cells of such chimeras. Moreover, lymph node cell suspensions and lymphoid tissue sections of neonatally thymectomized or adult thymectomized, irradiated and marrow-grafted mice contained an increased proportion of CRL. The results concur to establish proof that CRL are of extrathymic, bone marrow origin.     

Development of T and B cell areas in peripheral lymphoid organs of the rat

In the present study the early development of peripheral lymphoid organs (spleen, popliteal lymph node, mesenteric lymph node and Peyer's patches) is described in terms of homing patterns of T and B cells, demonstrated with immunohistoperoxidatic detection of characteristic membrane antigen in normal rats and with routine histology in neonatally thymectomized rats. In the first days after birth the peripheral lymphoid organs are almost exclusively populated by T cells. After neonatal thymectomy lymphocytes appear in the dome areas of Peyer's patches from four to six days after birth, in mesenteric and popliteal lymph nodes lymphocytes are found in the outer cortex from day 6 and day 8 respectively and in the marginal zone of the spleen from eight days onwards. These lymphocytes showed no membrane staining when reacted for T antigen with immunohistoperoxidatic techniques. The morphological evidence for considering Peyer's patches of rats as central inductive sites for the generation of B cells is poor. The discrepancy in the order of appearance of T and B cell (sub)populations in spleen compartments in normal ontogenetic development and lethally irradiated, stem cell reconstituted animals is discussed.     

Development of T and B cell areas in peripheral lymphoid organs of the rat.

In the present study the early development of peripheral lymphoid organs (spleen, popliteal lymph node, mesenteric lymph node and Peyer's patches) is described in terms of homing patterns of T and B cells, demonstrated with immunohistoperoxidatic detection of characteristic membrane antigen in normal rats and with routine histology in neonatally thymectomized rats. In the first days after birth the peripheral lymphoid organs are almost exclusively populated by T cells. After neonatal thymectomy lymphocytes appear in the dome areas of Peyer's patches from four to six days after birth, in mesenteric and popliteal lymph nodes lymphocytes are found in the outer cortex from day 6 and day 8 respectively and in the marginal zone of the spleen from eight days onwards. These lymphocytes showed no membrane staining when reacted for T antigen with immunohistoperoxidatic techniques. The morphological evidence for considering Peyer's patches of rats as central inductive sites for the generation of B cells is poor. The discrepancy in the order of appearance of T and B cell (sub)populations in spleen compartments in normal ontogenetic development and lethally irradiated, stem cell reconstituted animals is discussed.     

Characterization of Thy 1.2 positive cells in mouse bone marrow by sedimentation and mitogen stimulation.

By the use of unit gravity velocity sedimentation it was found that the majority of Thy 1.2 positive cells in the bone marrow of BALB/c mice sedimented in the same fractions as small lymphocytes of the marrow. This was shown both in normal, neonatally thymectomized and congenitally athymic mice. In all three groups of mice, two populations of Thy 1.2 positive cells were found. This indicates that these cells are cycling in the bone marrow. Long-lived T cells of normal bone marrow were included in the slowly sedimenting Thy 1.2 positive population (peak at 3 mm/h). Results after stimulation of bone-marrow cells with phytohaemagglutinin or concanavalin A indicated that the majority of Thy 1.2 positive cells in the bone marrow of thymus-deprived mice are effete end-products.     

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.     

Adult and pre-adult thymectomy of mice: contrasting effects on immune responsiveness, and on numbers of mitogen-responsive and Thy-1+ lymphocytes.

Peripheral lymphoid tissues of mice which have been thymectomized at 2 or 4 weeks of age, that is, before they achieve adult body weight, have been shown to be lacking in cells responsive to the T-cell mitogen, phytohaemagglutinin, when the animals became adult, and these mice have also been shown to have a deficient immune response against sheep erythrocytes. It is suggested these effects of pre-adult thymectomy are consequent upon removal of the prime source of T cells prior to the animal having acquired complete T-cell populations of the adult. Spleens and lymph nodes of mice thymectomized at 8 weeks of age were found to have reduced numbers of cells susceptible to the cytotoxic effects of anti-Thy-1 serum as early as 4 weeks after the operation, whereas the number of lymphocytes responsive to T-cell mitogens in these lymphoid tissues was not reduced at this time. The number of spleen-borne antibody-producing cells in a primary or secondary response was not affected by 8-week thymectomy, either when the response was tested in the operated animal, or after transfer of cells from such an animal to an irradiated recipient. The results are discussed with respect to other work on the effects of thymectomy of mice during the post-neonatal and pre-adult period.     

Delayed development of immunological responsiveness in neonatally thymectomized mice: a time-course study

Haemolysin responses to first injection of sheep erythrocytes in neonatally thymectomized, neonatally sham-thymectomized and intact Swiss albino mice were tested when the mice were 10 days, 4 weeks, 6–7 weeks and 6 months of age. The serum haemolysin activity was assessed at a number of times after injection of antigen (time-course study). Neonatal thymectomy of Swiss mice was followed by a decreased and delayed haemolysin response. These abnormalities in antibody response following neonatal thymectomy became less obvious when the age at which the mice were injected was increased, indicating that delayed development of immunological responsiveness had occurred in neonatally thymectomized Swiss mice.     

Kinetics of Theta-Positive and Theta-Negative Lymphocytes in Thymus-Deprived and Normal Mice

Life spans and proliferative kinetics of theta-positive (θ+) and theta-negative (θ-) cells were evaluated in normal, neonatally thymectomized (NeoTx) and sham-thymectomized (ShamTx) Balb/c mice. This was done by exposing cell suspensions, incubated with anti-θ antiserum + complement, to a dye exclusion test followed by fixation and autoradiography. Results from normal mice given [³H]thymidine injections 5 weeks before being killed indicated that long-lived θ+ and θ- cells constituted about equal percentages of the respective populations in peripheral lymphoid tissues. Long-lived θ+ cells constituted a relatively high percentage of θ+ cells in the bone marrow, whereas a minority of the Θ- lymphocytes were long-lived. Results from NeoTx and ShamTx mice, given intensive injections of [³H]thymidine, showed that the θ- cells of the mesenteric nodes were renewed at comparable rates in both groups of mice, whereas the θ+ cells were more rapidly renewed in NeoTx mice than in sham-operated littermates. In the bone marrow, the majority of both θ- and θ+ cells were very rapidly renewed, indicating a production of these cells in the bone marrow of ShamTx mice and also in NeoTx mice, in which the number of bone-marrow θ+ cells was significantly higher than in ShamTx littermates.     

Response of lymphocytes to plant lectins: I. A thymic-dependent lymphoid population responsive to Pokeweed mitogen

The response of lymph node, bone marrow, splenic and thymic lymphocytes to phytohaemagglutinin and Pokeweed mitogen has been studied. Depletion of theta positive or recirculating lymphocytes abolished the response to phytohaemagglutinin but did not impair the response to Pokeweed mitogen. Lymphocytes from thymectomized or athymic `nude' mice respond poorly or not at all to phytohaemagglutinin or Pokeweed mitogen.

We conclude that the normal response to Pokeweed mitogen depends upon an intact thymus gland. This despite the fact that one population of Pokeweed mitogen-responsive cells are theta-negative bone marrow-derived cells. This conclusion is based upon: (1) the equal reactivity of theta-positive and theta-negative lymphocytes to Pokeweed mitogen as shown by the normal response to Pokeweed mitogen of anti-theta serum treated lymphoid populations and (2) the impaired response of lymphocytes from nude mice and of lymphocytes from thymectomized, irradiated and bone marrow reconstituted mice to Pokeweed mitogen.

     

Selective depletion of lymphoid tissue by cyclophosphamide

Selective depletion of lymphocytes from the lymph follicles and cortico-medullary junction in lymph nodes and equivalent non thymus dependent areas of the spleen can be produced by cyclophosphamide (CY) (300 mg/kg) in the mouse and guinea-pig. Despite three such injections on alternate days, thymus dependent areas still contained lymphocytes. Total depletion of lymphocytes from lymph nodes and spleen was produced by combining neonatal thymectomy in the mouse or ALS treatment in the guinea-pig with CY. CY produced depletion of lymphocytes in the cortex of the thymus before the medulla. Maximal depletion occurred at 3 days and in surviving animals repopulation was evident by 7 days at the cortico-medullary junction only. Lymph follicles were found in lymph nodes of neonatally thymectomized CY treated mice following repopulation with bone marrow. These findings suggest that the lymphocytes of the lymph follicles are derived from a population of rapidly dividing cells, part of which at least can be found in the bone marrow.     

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.

     

Effect of thymectomy on polyoma tumor induction in CF 1 mice

Summary In CF1 mice thymectomized and inoculated with polyoma virus at birth, tumor formation was markedly accelerated; by 70 to 82 days after polyoma virus inoculation, tumors developed in 100% of neonatally thymectomized mice, while less than 20% of control mice developed tumors. Furthermore, grossly apparent polyoma tumors were produced in neonatally thymectomized CF1 mice when inoculated with polyoma virus at 21 days of age: of 18 neonatally thymectomized and polyoma virus inoculated-weanling mice, 3 were found to have grossly apparent tumors 100 to 150 days after the virus inoculation. No tumors were found in so treated sham-thymectomized mice, during the same observation period.Polyoma virus recovery from kidneys and antipolyoma virus antibody were almost equal in neonatally thymectomized mice and control mice. The data may indicate that anti-polyoma virus antibody has little influence on the production of polyoma tumors, and that neonatal thymectomy causes a higher incidence of polyoma tumors because of the reduced capacity of homograft immunity.By varying the days of operation and of virus inoculation after birth, it was found that operation two days after birth was as effective as thymectomy at birth, and inoculation of polyoma virus into neonatally thymectomized mice as late as 7 days after birth was as effective as inoculation at birth.The accelerating effect of neonatal thymectomy on polyoma tumor formation is suggestive that so operated mice may be used as hosts in search for oncogenic viruses.