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.     

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.     

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 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.     

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.     

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.

     

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.     

Effects of Anti-T Cell (Theta) and Anti-B Cell (Beta) Serum on the Immune Response in Mice

Heterologous anti-B cell (anti-beta) serum was prepared in rabbits against the spleen from neonatally thymectomized mice. The anti-beta serum, after absorption with thymus, is cytotoxic for bone marrow, bone marrow-derived cells, fetal liver and peritoneal lymphocytes. The cytotoxicity to the B cell can be absorbed out with bone marrow.

The cytotoxic effects of anti-beta serum on spleen and lymph node cells is compared to that of anti-theta serum. The data suggest that spleen has relatively more B than T cells, while lymph node has relatively more theta-positive cells.

To test the effect of anti-beta and anti-theta serum on the functional activity of lymphoid cells, C57 spleen or thymus was pre-incubated with the antiserum, in the presence of complement, and tested in vivo for graft-vs-host activity or transfer of an adoptive immune response to SRBC.

Treatment with anti-beta serum does not decrease the graft-vs-host activity of thymus or spleen cells. Anti-theta serum does decrease the graft-vs-host activity of both thymus and spleen cells.

Neither anti-beta serum nor anti-theta serum affect the phagocytic activity of peritoneal macrophages.

Both anti-beta serum and anti-theta serum decrease the transfer of an adoptive primary and secondary immune response to SRBC. A combination of anti-theta and anti-beta treated spleen can transfer adoptive immunity. Thymus and bone marrow can reconstitute the immunocompetence of anti-theta or anti-beta treated spleen respectively.

The results suggest that T cells alone can mount a graft-vs-host reaction and that this activity is not affected by anti-beta serum. The transfer of a humoral antibody response, on the other hand, requires functionally active T- and B-cells. This holds true for a primary as well as secondary immune response. Our anti-beta serum does not appear to have any anti-macrophage activity.     

Effects of Anti-T Cell (Theta) and Anti-B Cell (Beta) Serum on the Immune Response in Mice

Heterologous anti-B cell (anti-beta) serum was prepared in rabbits against the spleen from neonatally thymectomized mice. The anti-beta serum, after absorption with thymus, is cytotoxic for bone marrow, bone marrow-derived cells, fetal liver and peritoneal lymphocytes. The cytotoxicity to the B cell can be absorbed out with bone marrow.

The cytotoxic effects of anti-beta serum on spleen and lymph node cells is compared to that of anti-theta serum. The data suggest that spleen has relatively more B than T cells, while lymph node has relatively more theta-positive cells.

To test the effect of anti-beta and anti-theta serum on the functional activity of lymphoid cells, C57 spleen or thymus was pre-incubated with the antiserum, in the presence of complement, and tested in vivo for graft-vs-host activity or transfer of an adoptive immune response to SRBC.

Treatment with anti-beta serum does not decrease the graft-vs-host activity of thymus or spleen cells. Anti-theta serum does decrease the graft-vs-host activity of both thymus and spleen cells.

Neither anti-beta serum nor anti-theta serum affect the phagocytic activity of peritoneal macrophages.

Both anti-beta serum and anti-theta serum decrease the transfer of an adoptive primary and secondary immune response to SRBC. A combination of anti-theta and anti-beta treated spleen can transfer adoptive immunity. Thymus and bone marrow can reconstitute the immunocompetence of anti-theta or anti-beta treated spleen respectively.

The results suggest that T cells alone can mount a graft-vs-host reaction and that this activity is not affected by anti-beta serum. The transfer of a humoral antibody response, on the other hand, requires functionally active T- and B-cells. This holds true for a primary as well as secondary immune response. Our anti-beta serum does not appear to have any anti-macrophage activity.     

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.     

Enhanced primary resistance to Listeria monocytogens in T cell-deprived mice

The present studies reinvestigate the role of the T cell in the development of resistance to Listeria monocytogenes. Doubly thymus-deprived adult thymectomized irradiated bone marrow-reconstituted mice (D-AT × BM) were prepared using anti-theta serum-treated bone marrow from AT × BM donors for reconstitution. The growth of the bacteria in spleens and livers of D-AT × BM was inhibited and such animals survived infection with doses which were lethal to normal mice. Since the D-AT × BM animals were T cell-depleted, as evidenced by (a) absence of theta-positive cells in their spleens; (b) inability to mount a primary humoral response to a T-dependent antigen and (c) failure to reject H-2 incompatible skin allografts, their antibacterial resistance was not due to the presence of a residual T-cell population. Further evidence of T-cell depletion in these animals was furnished by the findings that, despite their resistance to L. monocytogenes, they failed to exhibit a delayed hypersensitivity reaction to Listeria antigens, their splenocytes were unable to transfer resistance to naïve hosts and they did not develop an anamnestic response upon secondary challenge.

We concluded from these findings that primary antibacterial resistance to L. monocytogenes need not necessarily depend on the development of specific cell-mediated immunity although under normal circumstances these two processes develop in chronological association. The increased resistance of D-AT × BM animals is interpreted as being due to the enhancement of bactericidal activity of mononuclear phagocytes, possibly caused by the removal of a regulatory T-cell population. This population seems to be radiosensitive and spleen-seeking and requires an intact spleen to mediate its effect.

     

Studies on thymus products. IV. Absence of serum `thymic activity'' in adult NZB and (NZB × NZW) F1 mice

New Zealand Black (NZB) mice and (B/W) F1 hybrids have a normal level of serum `thymic activity' (TA) at birth but this level decreases prematurely between the third and sixth weeks of life. At 2 months, NZB and NZ (B/W) F1 mice have no significant TA, whereas TA is still at birth's level in six control mouse strains and remains at this level until the fourth to the sixth month. Six weeks after the decline of serum TA, NZB mice show disappearance of theta-positive lymph node rosette-forming cells (RFC) and 2 weeks later, progressive decrease in spleen RFC sensitivity to anti-theta serum (AΘS) and azathioprine (AZ) as in neonatally thymectomized CBA mice. Normal AZ and AΘS sensitivity of spleen and lymph node RFC is reconstituted by in vitro or in vivo treatment by thymic extracts. The early thymic abnormalities found in NZB mice are in keeping with the thymic medullar epithelium atrophy reported in newborn NZB mice.     

The effect of antigen deprivation on thymus-dependent and thymus-independent lymphocytes in the small intestine of the mouse

Isografts of foetal small intestine, implanted under the kidney capsules of adult mice grow normally and, despite the lack of intraluminal antigenic stimulation, are populated by thymus-dependent and thymus-independent lymphocytes. The Peyer's patches in these grafts are very small, lack germinal centres and can be shown to have small thymus-dependent areas.

Quantitative intraepithelial lymphocyte counts were carried out in normally sited small intestine of immunologically intact and thymus-deprived mice, and in grafts implanted in intact and thymus-deprived mice. Counts were also performed in a group of neonatally thymectomized and control mice. The results show a significant depletion of intraepithelial lymphocytes in neonatally thymectomized mice, and a profound reduction in numbers of intraepithelial lymphocytes in grafts, deprived of antigen, when compared with normally sited intestine of the same age.

     

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.     

Significance of T cells in resistance to experimental murine coccidioidomycosis.

The resistance of mice to coccidioidomycosis was found to be dependent on lymphoid cells. Thus, spleen cells from mice immunized with killed spherules of Coccidioides immitis, when transferred to irradiated (500 R) recipients, conferred upon the recipient mice resistance to infection with C. immitis. Prior incubation of these spleen cells with anti-theta serum in the presence of complement abrogated their capacity to protect the recipients from infection with C. immitis. Adult thymectomized mice, which had been irradiated (800 R) and reconstituted with bone marrow from normal mice, were more susceptible to infection with arthrospores than were nonthymectomized, irradiated bone marrow-reconstituted controls. Genetically homozygous athymic ("nude") mice died after infection with a dose of arthrospores that was sublethal for their heterozygote counterparts possessing a thymus, or for normal mice. The results indicate that a functioning T-cell population is an essential component for effective immunity to C. immitis.     

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.     

The bursal origin of an immunocompetent cell for antibody formation in the chicken.

Surgically bursectomized and irradiated chickens were given bursal, splenic, bone marrow or thymic cells taken from syngeneic donors, together with killed Brucella abortus and Salmonella pullorum. Blood samples were taken from those chickens 7 days later, and the serum agglutinin titres were determined. The cells of any lymphoid organ taken from 28-day-old chickens were more effective in restoring antibody response than those from 18-day-old ones. The restorative capacities of the bursa and splenic cells were greater than those of the bone marrow and thymic cells. On the other hand, splenic, bone marrow or thymic cells taken from bursa-less chickens, and lymphoid cells taken from normal chickens but treated with anti-bursa serum in the persence of complement, were virtually incapable of restoring the immune response. Bursal, splenic or bone marrow cells taken from neonatally thymectomized chickens, and bursal or thymic cells treated with anti-thymus serum were effective, being comparable with the corresponding cells taken from normal chickens or treated with normal sera, in restoring the suppressed immune response in chickens devoid of the lymphoid system. These facts clearly indicate that the primary and central agent crucial for development of the humoral immune response against the two bacterial antigens tested is the bursa. It is strongly suggested from the results of adoptive immunization using intrabursally primed cells that the cells recognizing Brucella abortus exist within the bursa of 4-day-old chicken.     

Infectious immunological tolerance

Previous studies have shown that thymectomized lethally irradiated bone marrow grafted mice, reconstituted with thymocytes and pretreated with a large dose of sheep red blood cells (SRBC), are unable to respond to a subsequent immunizing injection of SRBC even after an inoculation of normal thymocytes. If, however, the mice are not thymocyte reconstituted prior to the pretreatment with SRBC, they can respond almost normally to an immunizing injection of SRBC if inoculated with normal thymocytes after the termination of antigen pretreatment.

In the present study the immunosuppressive effect of the presence of thymocytes during the antigen pretreatment was studied by adoptively transferring the spleen cells of the antigen pretreated mice to thymus-deprived chimeras. These spleen cells not only did not co-operate with normal thymocytes in the secondary hosts, but they also prevented the co-operation of normal thymocytes with normal bone marrow derived cells. Untreated spleen cells or treated spleen cells from mice not reconstituted with thymocytes did not affect cell co-operation in the secondary hosts. The abrogation of the co-operation in the secondary host was specific in that the addition of spleen cells did not affect the anti-horse red blood cell response. If the primary host made antibody as a result of the pretreatment, the transfer of their spleen cells did not prevent antibody production in the secondary host.

     

The development of NK cell activity in thymectomized bone marrow chimaeras.

Most of the natural killer (NK) cells, recently derived from the bone marrow (14 days after injection of bone marrow cells into lethally irradiated mice), express the Thy-1 antigen; after some period of time (by day 28) a normal proportion (50%) of Thy-1+ NK cells is found. This study demonstrates that the shift of these Thy-1+ NK cells to Thy-1- NK cells is influenced by the thymus: NK cells developing in the total absence of the thymus--bone marrow (bm) cells from adult thymectomized (Tx) or nude (nu) mice injected into thymectomized recipients, (Tx) bm----(Tx) or nu bm----(Tx)--remain Thy-1+, while the presence of the thymus at some stage of the NK cell maturation--bone marrow cells injected into thymectomized recipients, bm----(Tx), or normal recipients reconstituted with bone marrow cells from thymectomized or nude mice, (Tx) bm----or nu bm----normal recipients--is sufficient for the development of normal ratio of Thy-1+ and Thy-1- NK cells.     

Studies on thymus products: II. Demonstration and characterization of a circulating thymic hormone

Normal mouse serum confers on bone marrow rosette-forming cells (RFC) from normal mice a high sensitivity to anti-theta serum (AθS) and azathioprine (AZ) which they otherwise lack. Such an effect can also be demonstrated on spleen cells from adult thymectomized mice, `thymus-deprived' and nude mice, but the amount of serum required is higher in the latter mice. This activity of serum on RFC disappears after thymectomy of the serum donor with a half-life of 2.5 hours and reappears in thymectomized mice within 4 days after grafting of a thymus. Serum thymic activity (TA) is present in different amounts in different mouse strains and in ageing mice it progressively disappears. No TA is found in the serum of 4-week-old nude mice. TA is stable after lyophilization but is thermolabile in solution. It passes through UM 10 Amicon membranes, which suggests that its molecular weight (mol. wt) is probably < 10,000. TA is reversibly suppressed in the presence of a serum inhibitor with a mol. wt between 100,000 and 300,000. This inhibitor is not detectable in the serum of thymectomized mice unless the serum is incubated with TA containing serum for 60 minutes at 37°. The biological significance of TA is still a matter of speculation but its role in maturation or expansion of T-cells is suspected.