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.     

The effect of pertussis adjuvant on antibody production: the need for thymus-dependent lymphocytes

The response to human serum albumin in the BALB/c mouse immunoglobulin classes has been examined both with and without B. pertussis (BP) organisms as adjuvant. Without BP, IgG1 was found to be the only thymus-dependent class. The presence of thymus-dependent lymphocytes was necessary for BP to have its full adjuvant effect, although some activity was observed in neonatally thymectomized mice. In thymectomized mice IgG1 antibody was stimulated significantly by BP, but only to half the level seen in intact mice. Although IgG2a and IgG2b both rose in these mice after BP treatment, the difference was not significant. IgA and IgM were both unchanged. Thus, as well as being needed for increased stimulation of IgG1, the thymus was particularly required for BP to increase the response in the classes of immunoglobulin not usually found to be thymus-dependent for soluble antigens.     

Differentation of foetal mouse thymus. Ultrastructure of organ cultures and of subcapsular grafts

Thymuses removed from 12–13-day-old foetal (C57×CBA)F₁ mice, were grown in organ culture for up to 25 days. Initially the thymuses consisted predominantly of undifferentiated epithelial cells and some large lymphoblasts. The epithelial cells differentiated rapidly but epithelial cells in mitosis were seen throughout the entire culture period. The lymphoblasts, however, showed a restricted period of proliferation between the 2nd and 10th days of culture which resulted in the production of large numbers of typical small lymphocytes. Many of these cells died in situ but even after 25 days many apparently viable small lymphocytes were present.

Some 14-day cultures were implanted beneath the kidney capsule of young neonatally thymectomized or sham thymectomized syngeneic mice. These grafts lost their complement of small lymphocytes within the first 24 hours but their epithelial cells rapidly started to proliferate and the grafts became infiltrated with large lymphoblasts. After 1 week a small thymus was reformed and after 6–10 weeks up to 50 mg of thymic tissue could be obtained from each graft. Electron microscopy showed that the cortex of the graft was indistinguishable from a normal thymus but the medulla was small and contained few characteristic medullary epithelial cells.

     

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.     

The effect of erythrocyte antigen structure on requirement for T cells

The induction in mice of a humoral immune response to intact sheep erythrocytes, both in vivo and in vitro, requires participation of thymus-derived (T) lymphocytes. In an in vitro system, spleen cells from both neonatally thymectomized and adult thymectomized irradiated bone marrow protected mice were successfully immunized, using washed sonicated sheep erythrocyte membrane fragments as antigen. This obviation of the requirement of T lymphocytes in the immune response, coupled with previous work on macrophage independence, indicates that sonicated membrane fragments were capable of directly immunizing bone marrow-derived (B) lymphocytes in vitro.

These results further confirm the signal importance of antigenic structure in determining the cellular requirements for an immunological response; whereas antigens of particulate or monomeric form require the presence of both T cells and macrophages, polymeric antigens of intermediate size such as polymerized flagellin and sonicated sheep erythrocyte membranes require neither of these accessory cells.

The results caution against the use of erythrocytes as models of thymus-dependent antigens. The data further suggest that reports of late antibody responses of relatively normal magnitude in thymectomized animals given larger doses of heterologous erythrocytes may have been due to direct immunization of B lymphocytes by degraded erythrocyte antigen.

     

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.     

Secretion of IgA into 'antigen-free' isografts of mouse small intestine

The immunoglobulins secreted into "antigen-free" isografts of mouse small intestine have been measured by single radial immunodiffusion and immunoelectrophoresis of tissue extracts. IgA was detected in high concentration, and small amounts of IgG1 and IgG2 were also present. The IgA content of material within the graft lumen was considerably higher than the IgA content of the graft wall, indicating that IgA had been secreted and stored. Graft IgA increased with time after implantation, and no differences in immunoglobulin contents were found when grafts in thymus-deprived and in normal mice were compared. A group of host mice had been immunized with BSA and had high titres of circulating antibody to BSA; anti-BSA was not detected in the grafts implanted in these mice. However, graft extracts had moderately high titres of bacterial agglutinins when tested against a panel of commensal gut bacteria.

These results indicate that secretion of IgA into a segment of small intestine is not dependent upon the presence of antigens within its lumen; however, the immunoglobulins secreted may have antibody activity against antigens present in small intestine elsewhere in the animal.

     

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.     

Experimental cutaneous leishmaniasis. III. Effects of thymectomy on the course of infection of CBA mice with leishmania tropica

This paper describes the course of infection and the immunological response in thymus-deprived and normal CBA mice after intradermal inoculation with promastigotes of L. tropica.

Infection of normal mice resulted in the development of a cutaneous ulcer healing within 12 weeks. As the infection progressed and the draining lymph nodes increased in weight, changes in the paracortical and follicular regions were accompanied by the development of delayed hypersensitivity and the production of antibodies detectable by immunofluorescence and a parasite agglutination test.

Lesions in thymectomized irradiated mice healed more slowly and the draining lymph nodes were smaller than in normal mice. Follicular reactions were feeble and paracortical activity depressed. The most noticeable feature of these lymph nodes was a persistent and intense macrophage infiltration. Delayed hypersensitivity and antibodies detectable by immunofluorescence were correspondingly low; but parasite agglutinating antibody was not depressed. The course of infection and immunological response of a control group of sham thymectomized, irradiated mice resembled that of normal mice.

These experiments indicate that thymus-dependent cell populations play an important role in the response of mice to infection with L. tropica.

     

Qualitative and quantitative morphologic study of peyer's patches of the mouse after neonatal thymectomy and hydrocortisone injection

Peyer's patches in normal adult mice, neonatally thymectomized mice and mice injected with hydrocortisone were studied qualitatively and quantitatively by light microscopy. The patch was divided into germinal center, follicular area, parafollicular area and dome area. In normal mice, the volumetric ratio of the germinal center to the entire patch was 30.9%; that of the follicular area, 33.3%; that of the parafollicular area, 27.7%; and that of the dome area, 8.2%. Thymus-dependent small lymphocytes were 40% of small lymphocytes in the patch. Out of the total thymus-dependent small lymphocytes in the patch, 13% were included in the germinal center; 19%, in the follicular area; 62%, in the parafollicular area; and 6%, in the dome area. Hydrocortisone-sensitive small lymphocytes were 65% of the total small lymphocytes in the patch, the germinal center contained 9%; the follicular area, 84%; the parafollicular area, 2%; and the dome area, 5%. The epithelium over the dome area was invaded by numerous small lymphocytes. Forty-eight percent of lymphocytes within the epithelium over the dome were thymus-dependent and 67% were hydrocortisone-sensitive. It is concluded that Peyer's patch may be considered as a peripheral lymphatic tissue, functionally as well as morphologically.     

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.     

Lymphocyte proliferation in neonatally thymectomized rats

Autoradiography has been used to evaluate lymphocyte proliferation in the neonatally thymectomized rat in comparison with the normal animal. The data obained show that the proliferative activity of lymphocytes is greatly increased in the thymus-dependent areas 4–6 weeks after thymectomy, whereas it is normal or slightly increased 3 months laters. It seems plausible to assume that a thymus factor or chalones in situ produced by specific cells normally regulate the proliferation of thymus-derived cells. The increase of the proliferative activity accounts for the repopulation of the thymus-dependent areas, which are completely replenished in the older animals. Recirculation of the thymus cells is not confined to the thymus-dependent areas.     

Selective migration of lymphocytes within the mouse small intestine

The factors which determine the migration of lymphoid cells to lamina propria or Peyer's patches of mouse small intestine have been investigated by autoradiographic tracing of intravenously injected spleen, thymus and lymph node cells. The numbers of labelled cells found in antigen-free grafts of foetal small intestine were compared with the numbers in normally sited gut. Thymus, normal spleen and B spleen lymphocytes, labelled with [³H]adenosine or [5-³H]uridine, were confined to Peyer's patches in normal and grafted gut. [³H]Thymidine-labelled lymphoblasts from the mesenteric nodes of young (19–22 days) mice and mice infected with Nippostrongylus brasiliensis were found in the lamina propria of both graft and normal small intestine, but [³H]thymidine-labelled lymphoblasts from oxazolone-primed lymph nodes did not migrate to the villi. The possible roles of intraluminal antigens, source of cells and changes in cell surface receptors during differentiation, in determining the selective migration of cells to the lamina propria and Peyer's patches, are discussed.     

Studies on thymus products: I. Modification of rosette-forming cells by thymic extracts. Determination of the target RFC sub-population

Thymic extracts confer on normal bone marrow rosette-forming cells (RFC) in vitro a high sensitivity to anti-theta serum (AθS) and azathioprine (AZ) which they usually lack. Thymic extracts can also confer high sensitivity to AθS and AZ to spleen RFC from adult thymectomized, neonatally thymectomized `thymus-deprived' and nude mice. However, the amount of thymic extracts necessary to get the effect is significantly higher on thymus-deprived and nude mouse spleen RFC than on RFC from spleens of adult thymectomized mice or normal mouse bone marrow. Thymic extracts are also active in vivo and there is a good correlation between the in vitro minimum concentration giving AθS and AZ sensitivity to spleen RFC from adult thymectomized mice and the in vivo minimum dose giving such an effect after intravenous injection. The effect of extract in vivo does not appear before the fourth hour after the injection and is transient, disappearing after 48 hours. Injection of thymic extracts induces the appearance of a `thymic activity' (TA) in the serum with a short half-life (2 hours). Injection of thymic extracts into normal mice does not modify sRFC characteristics in spleen, lymph nodes and bone marrow. It is suggested that thymic extracts act reversibly on a population of T-RFC precursors.     

The persistence of donor-derived cells in thymus grafts, lymph nodes and spleens of recipient mice

Thymus was labelled in vivo by injecting newborn C3H/Bi mice with three or four doses of 2 μc of [³H]thymidine. The labelled thymus was grafted into the kidney capsule of 22–25-day-old intact and thymectomized syngeneic recipients which were killed 18, 41 or 42 days later. The grafts and lymphoid tissue were sectioned and examined by autoradiography after exposure times of up to 26 weeks.

Small and medium labelled lymphocytes were seen in the thymus-dependent areas of the lymph nodes and spleen of all recipients provided the sections were exposed for a sufficiently long period. More labelled cells were seen in recipients killed at 18 days than at 41 or 42 days after grafting. It was concluded that these cells were the direct descendants of those cells labelled in the graft. There was no evidence of re-utilization of the isotope label.

Most of the cells originally labelled in the graft either lost their label by multiple divisions or were replaced by unlabelled cells; although the epithelial-reticular cells remained heavily labelled. The importance of this last finding is discussed.

     

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.

     

The failure to transfer the NZB disease following implantation of NZB thymus-containing chambers

The role of the thymus in the disease of the NZB still remains to be clearly defined. Upon the basis of Coombs' positive reactions occurring in normal strains of mice following the implantation of thymus-containing diffusion chambers, previous workers suggested the primary role of a subcellular thymus causal factor in the disease of the NZB. We have been unable to confirm these findings in neonatally and adult thymectomized or intact mice of two other strains in spite of being implanted with NZB thymus-containing chambers.     

Recovery of immunological responsiveness in thymectomized mice

After a limited period of immunological unresponsiveness, neonatally thymectomized colony-bred Swiss mice were found to recover their ability to form haemagglutinins and haemolysins as well as their antibody-plaque-forming capacity following injection of sheep erythrocytes. No such spontaneous reconstitution was observed in F₁-hybrids of highly inbred CBA and CBA-T6T6 mice. Adult thymectomized and irradiated Swiss mice similarly regained their ability to form haemolysins and haemagglutinins, but no regeneration of antibody-plaque production occurred in these mice during the period of observation. No regular correlation was found between the degree of immunological deficiency on the one hand and the level of circulating lymphocytes or the histological appearance of the spleens on the other, following neonatal thymectomy or adult thymectomy and irradiation.

The possible mechanism of recovery from immunological impairment after thymectomy and the apparent discrepancies between overall haemolysin production and haemolytic plaque production in the spleen are discussed.

     

Hormones and the immunological capacity. V. Modification of growth hormone-producing cells in the adenohypophysis of neonatally thymectomized germ-free mice: an electron microscopical study

An electron microscopical study has been carried out to evaluate the effect of neonatal thymectomy on the hypophysis of germ-free mice at different times after the operation. The results fully confirm the previous findings in neonatally thymectomized, conventional mice. Also neonatal thymectomy in germ-free mice results in degranulation of growth hormone-producing cells in the anterior pituitary gland. A large number of these cells show an enlarged endoplasmic reticulum with formation of cisternae and loss of hormone granuli. This alteration of growth hormone-producing cells is similar to that observed in other cells of the hypophysis after removal of other target glands such as thyroid or gonads. The changes in the growth hormone-producing cells in neonatally thymectomized germ-free mice occur even in the first days after birth, when the number of differentiated growth hormone-producing cells is still very low.

Some thymectomized germ-free mice showed symptoms of the wasting syndrome but the alterations in their hypophysis were not more pronounced than those observed in thymectomized germ-free but nonwasting mice. The data fit well our suggestion that the perinatal thymus is under hypophysial control and that immunological maturation depends on endocrine function.