Abnormal stem cells in autoimmune-prone mice are responsible for premature thymic involution

Autoimmune-prone mice show premature thymic involution, including morphological and functional abnormalities. To determine why the thymic abnormalities develop in autoimmune-prone mice, transplantation of the thymus and/or bone marrow was performed. When thymuses of newborn MRL/1 (H-2k) mice were grafted into C3H/HeN nu/nu(H-2k) mice, the engrafted thymuses did not show the abnormalities which characterize the thymus in the autoimmune-prone MRL/1 mice. By contrast, when thymuses of newborn C3H/HeN or MRL/n mice were grafted into MRL/1 mice, the engrafted thymuses developed after an interval of 3 months the same morphological abnormalities as were seen in MRL/1 mice. Thus, we can conclude that premature involution of the thymus in autoimmune-prone mice may not be a genetically determined abnormality intrinsic to the thymus, but rather an abnormality secondary to other events occurring in these mice. When bone marrow of young C3H/HeN nu/nu mice was transplanted into irradiated (850 rad) MRL/1 mice, neither thymic abnormalities nor autoimmune diseases developed. Therefore, it seem likely that abnormal stem cells in autoimmune-prone mice induce thymic abnormalities, and these, in turn, are associated with the development of autoimmune diseases.     

Induction of immunological tolerance by chimeric thymus transplantation

The role of bone marrow derived cells (BMD) within the thymus during the induction of immunological tolerance was investigated using transplantation of chimeric thymuses. Chimeric thymuses were constructed by reconstituting lethally irradiated BALB/C recipients with T-cell depleted C3H bone marrow (BM) cells. Two to three months post bone marrow transplantation the thymuses of these animals, in which the epithelium was of BALB/C origin and BMD cells of C3H origin (verified by immunoperoxidase staining), were transplanted into thymectomized, lethally irradiated BALB/C recipients which were reconstituted with T-cell depleted syngeneic (BALB/C) bone marrow cells. Induction of specific tolerance to the BMD cells (C3H origin) present in the chimeric thymus could be demonstrated in MLR at 3 to 6 months post chimeric thymus transplantation. It is concluded that bone marrow derived cells are able to induce immunological tolerance within the thymus.     

The effect of sequential multiple grafting of syngeneic newborn thymus on the immune functions and life expectancy of aging mice

Enhancement of the immune functions and extension of the mean life expectancy were successfully performed in aging mice by sequential multiple grafting of syngeneic newborn thymus. In the first experiment, 2-month-old female C57BL/6 mice were grafted with either syngeneic newborn thymus or newborn spleen every 2 months, 5 or 6 times. A significant enhancement of T cell dependent immune functions were observed in the group sequentially grafted with newborn thymus, in comparison to that grafted with multiple sequential newborn spleen or with a single newborn thymus and that without a graft. In the second experiment, the same sequential grafting protocol was performed in middle aged mice at monthly interval for 4-5 consecutive months and the immune functions and survival rate were compared between the experimental and control groups. The immune functions were only partially rejuvenated, but an extension of the mean remaining life expectancy was observed in the experimental group (312 +/- 38 days) as compared with control (214 +/- 42 days), although maximal life-span was the same in both groups (1100 days).     

Age-dependent separation of class-specific suppressor cells in thymus of SJL/J mice

The thymus of SJL/J mice of age 3-6 weeks has been previously shown to contain suppressor cells that inhibit the antibody response to lymph node cells to SRBC. The effect of these suppressor cells disappear as the animals age (24 weeks or more). We find that these aged animals acquire thymic suppressor cells which suppress the generation of cytotoxic T-cells both in vitro and in vivo. Although such suppressors are not present in the thymuses of young SJL/J mice, suppression can be induced by treatment with estrogen and progesterone. The differentiation of functionally different suppressor cell populations in thymus may be affected by both age and hormonal status.     

Immunological status of nude mice engrafted with allogeneic or syngeneic thymuses

Restoration of T-cell functions and changes in autoantibody production were studied in BALB/c nu/nu (nude) mice engrafted with syngeneic (BALB/c) or allogeneic (C57BL/6J) thymuses across major histocompatability barriers. T-cell functions, including mitogen responses and antibody production to sheep red blood cells (SRBC), were restored in nude mice engrafted with either allogeneic or syngeneic thymuses. Alloreactivity was evaluated by analysis of the pattern of skin allograft rejection, generation of alloreactive cytotoxic T-lymphocytes (CTLs), or quantitation of mixed-lymphocyte reaction (MLR). BABL/c nude mice engrafted with thymuses from newborn C57BL/6J mice accepted the skin from either thymus donor-type mice or from host-type mice. By contrast, such thymic chimeras rejected skin grafts from a third-party donor. CTLs from nude mice engrafted with C57BL/6J thymuses were cytotoxic to target cells of the third party but not to target cells of the host-type or of the thymus-type. In the MLR assay, spleen cells of nude mice engrafted with C57BL/6J thymuses responded vigorously to third party cells and only slightly to cells of the thymus-type. Low levels of serum IgG and high titers of IgM antibodies to nuclear antigens (but not dsDNA) or skin basal cells were also found in nude mice. Antibodies to both nuclear antigens and skin basal cells disappeared after transplantation of syngeneic thymuses, but not after transplantation of allogeneic thymuses. By contrast, serum IgG levels were restored to normal in nude mice engrafted with either syngeneic or allogeneic thymuses. These results suggest that either HLA-matched or HLA-mismatched thymus grafts may become a viable treatment for certain patients with T cell deficiencies associated with deficient development or maintenance of thymic structure and/or function.     

Idiotype regulation of thymus autoantibodies.

Rabbit antisera specific for idiotypic determinants (Id) of monoclonal Thy-1 autoantibodies were tested for their capacity to elicit Id-bearing thymus autoantibodies in mice. Rabbits were immunized with the 20-10-5 monoclonal IgM Thy-1 autoantibody, and idiotype-specific antibodies (anti-Id) were obtained by affinity chromatography. BALB/c and C3H mice were immunized by repeated i.p. injections of the anti-Id reagent. Control mice received repeated injections of purified normal rabbit immunoglobulin (NRIg) sheep red blood cells (SRBC) or human serum albumin (HSA). The sera of the treated mice were examined for their reactivity with anti-Id xenoantisera and for their capacity to react with thymus cells. The injection of anti-Id sera into either BALB/c or C3H mice resulted in a significant increase in the serum concentration of Id-bearing immunoglobulins, while no such change was observed in the sera of mice injected with NRIg. The administration of anti-Id, NRIg, SRBC or HSA resulted in a gradual increase in the concentration of autoantibodies reactive with thymus cells. Incubation with anti-Id sera prevented the binding to thymus cells of autoantibodies induced by anti-Id treatment. In contrast, the binding capacity of autoantibodies induced by NRIg, SRBC or HSA was unaffected by anti-Id sera. Thus, only the thymus autoantibodies induced by anti-Id treatment expressed Id-determinants. These results demonstrate the existence of a regulatory network in the formation of thymus autoantibodies.     

Radiation-induced-thymic lymphoma occurs in young,but not in old mice

Young mice exposed to fractionated whole-body irradiation develop thymic lymphoma. By using young and old mice, we examined the effect of age on the occurrence of radiation-induced thymic lymphoma in mice. In the first experiment, young and old mice were grafted with newborn thymus under kidney capsule and then treated with fractionated whole-body irradiation (FWI). In the second and third experiments, four combinations of bone marrow chimeras were constructed by transplanting bone marrow cells from young and old mice into young and old mice. Then these chimera mice were grafted with newborn thymus and treated with fractionated whole-body irradiation. The results in the present study indicate that the incidence of thymic lymphoma is influenced by age factors of thymic microenvironment, bone marrow, and host environment. If they are all young, the incidence of thymic lymphoma is high. If one of these is old, the incidence definitely decreases. Thymic lymphoma never occurred in old thymic environment even in the presence of young thymocytes. In conclusion, age advantage is present in the induction of thymic lymphoma after the treatment with FWI and the incidence definitely decreases in the presence of old factors.     

Limiting dilution analysis of alloantigen-reactive T lymphocytes. VI. Ontogeny of cytolytic T lymphocyte precursors in the thymus

The ontogeny of development of precursors of cytolytic T lymphocytes (CTL-P) in the murine thymus has been investigated. Fetal or newborn thymocytes harvested from C57BL/6 mice were cultured with irradiated allogeneic (DBA/2) spleen cells in limiting dilution mixed leukocyte microcultures and assayed for cytotoxicity against 51Cr-labeled P-815 (DBA/2) tumor target cells. By applying Poisson statistics to these data, minimal estimates of thymic CTL-P frequencies were determined. Whereas CTL-P were barely detectable in fetal or newborn thymuses (3-19 CTL-P/10(6) cells), their frequency increased dramatically (to 44-127 10(6) cells) within 24 h of birth. In comparison, control adult (day 70) thymuses contained 300-400 CTL-P/10(6) cells. Mixing experiments indicated that the low CTL-P frequencies observed in newborn thymuses could not be attributed to a suppressive phenomenon. These data provide strong indirect evidence for functional maturation of CTL-P within the neonatal thymus.     

Age-related polymorphism of thymus subpopulations in inbred mice.

There are striking age-related changes in the demography of thymus lymphocytes, i.e. in thymus-cell subpopulations of BALB/c and SJL mice; these changes occur in the proportion of cells, identified by various markers, and by the membrane density of these markers. The thymuses of both strains undergo an age-related increase in the proportion of CD4+ CD8- cells and decrease in CD4+ CD8+ cells. Age-related changes in cells that are Pgp-1+ also show marked strain differences: Pgp-1+ cells increase in SJL, but not in BALB/c thymuses. In both strains, cells with high density of Pgp-1 appear in later life, though this is more marked in the thymus of SJL, which also shows a higher relative density at an advanced age, than do BALB/c mice. Furthermore, the per cent of cells with high density of Pgp-1 is larger in thymuses of SJL than in BALB/c mice. The percentage of CD45+ thymocytes remains unchanged, as animals age. Thymocyte-membrane densities of CD-45 undergo age-related increases in both SJL and BALB/c. The per cent of cells with high density of CD-45 is similar in both strains. Individual variations in relative size of subpopulations in SJL mice of the same age are greater in old than in young mice; this increase in heterogeneity is manifested by increase in standard deviation. Corresponding significant changes have not been observed in BALB/c or C57BL/6 mice. Thus, we have detected an intrastrain variation which may reflect age-related effects of the impact of stochastic events.     

The initial characterization of a thymus factor chemotactic to bone marrow cells

Thymus supernatants were produced by cluturing minced newborn CBA/J mouse thymuses in serum-free media for 48 h. Supernatants thus obtained were chemotactic to a subset of bone marrow cells as assessed in blind well chambers, and enriched for immature lymphoid cells in the migrating cell population. The enriched population of cells was shown to be capable of homing to the thymus of an irradiated mouse in vivo in a significantly higher percentage than nonmigrated bone marrow cells. In this report, initial characterization of the factor(s) responsible for this in vitro migration is presented. Several well studied thymic factors were compared with the thymus supernatants for their ability to induce migration of bone marrow cells in vitro. Thymulin (FTS-Zn), FTS, and TP-5 (the pentapeptide fragment of thymopoietin) were used. None of these factors demonstrated chemotactic properties in the migration assay using concentration ranges in which other in vitro activities have been observed. The chemoattractive activity of the supernatant was unaltered by ultracentrifugation. The effects of temperature on the chemotactic properties of thymus supernatant were examined, and a fifty percent decrease in observed migration occurred with thymus supernatant heated to 100 degrees C for 1 h. In addition, incubation of the supernatant for 1 h at 37 degrees C with chymotrypsin, but not with trypsin, inhibited migration, presumably by inactivation of the active factor. Using Amicon microconcentrators, the supernatant was separated into several fractions based on molecular weight. Initial data suggest that the active fraction is in the less than 10,000 mw range.     

Strain differences in the immune response of mice: III. A raised tolerance threshold in NZB thymus cells

Irradiated (NZB×BALB/c)F1 mice were injected with syngeneic bone marrow cells, syngeneic or parental thymus cells, and sheep red cells. The antibody plaque-forming cell response depended on the number of sheep cells and the age and strain of the thymus cells. Young or adult BALB/c thymus, and young hybrid thymus, responded best to low numbers of sheep cells; with higher numbers they became tolerant. Adult hybrid thymus, and young or adult NZB thymus, responded better to high numbers of sheep cells. Hybrid mice irradiated and restored with BALB/c bone marrow developed thymus cells with the reactivity of BALB/c thymus. It is argued that NZB mice, and older hybrids, may develop autoimmunity because of an abnormality of tolerance induction manifested in their thymus cells, but of bone marrow origin.     

RNA synthesis in the thymus of the immunologically mature mouse

RNA synthesis was investigated in the thymus glands of adult immunized mice. After the intraperitoneal injection of mice with sheep red blood cells (SRBC), the net synthesis of RNA in the gland increased. A small but consistent amount of the RNA synthesized was distinguished by RNA:DNA hybridizations from that found in the glands of mice not injected with antigen. The RNA formed after immunization did not appear by hybridization to be specific for different antigens since the species of RNA formed in the glands of mice injected with SRBC was indistinguishable from RNA formed in the thymuses of mice injected with chicken red blood cells. RNA synthesized in the thymus glands of mice pharmacologically `stressed' by the injections of hydrocortisone, however, was distinguishable from that formed in the glands of mice injected with antigen.     

Induction of a T-cell specific antigen on bone marrow lymphocytes with thymus RNA.

Expression of a rabbit T-cell specific antigen can be induced on bone marrow lymphocytes following exposure to an RNA extract obtained from the thymuses of young rabbits. The presence of the antigen was demonstrated using goat anti-rabbit T-cell serum in a complement-dependent cytotoxicity assay. The T-cell antigen first appeared 3 h after addition of the thymus RNA to bone marrow cell cultures and the maximum number of cells expressing the T-cell antigen was observed within 24 h. RNA obtained from a source other than the thymus was found to be ineffective in inducing expression of the T-cell antigen. The induction of the antigen appears to be dependent on the presence of intact thymus RNA, as RNase treatment but not trypsin treatment, destroyed the ability of the RNA to induce the T-cell antigen.     

Restraint and sound stress reduce the in vitro migration of prethymic stem cells to thymus supernatant.

Murine bone marrow cells were examined in an in vitro assay to determine whether stress modulates the migration of prethymic stem cells to thymus supernatant. Adult CBA/J mice were either restraint or sound stressed for two hours daily for five days. Bone marrow cells were removed and migrated toward newborn thymus supernatant in an in vitro migration assay in blind well chambers. Bone marrow cells from animals which had been stressed for five days showed a significant decrease in the percent migration to thymus supernatant when compared to bone marrow cells from age-matched control mice. This suggests that either a smaller proportion of precursor cells are available in the bone marrow for migration to the thymus or the number of cells remains the same but these cells are less responsive to chemoattractive factors in the supernatant, thus causing them to migrate at a decreased rate.     

Traffic of peripheral B and T lymphocytes to hyperplastic, preneoplastic thymuses of AKR mice.

AKR mice develop hyperplasia of the thymus before the development of retrovirus-associated lymphoma at that site. This hyperplasia, first detectable in AKR/J mice by 4 weeks of age and in AKR/C mice by 4 to 5 months of age, is characterized by an enlarged thymic medulla that contains T and B lymphocytes. In contrast to the general population of thymocytes, most of these T and B lymphocytes have a mature immunophenotype that includes expression of high levels of the MEL-14-defined (gp90) 'homing receptor' for peripheral lymph node high endothelial venules. In vivo homing studies reveal a marked increase in traffic of peripheral lymphocytes (T more than B) to the hyperplastic thymuses of old AKR mice as compared to histologically normal thymuses of age-matched BALB/c and C57BL/Ka mice or young AKR mice. These changes correlate chronologically with changes in retrovirus antigen expression in AKR thymuses and suggest a role for the traffic of lymphocytes from the periphery to the thymus in response to local antigenic stimulation in the pathogenesis of thymic hyperplasia in AKR mice.     

Role of pre-T cells and chemoattractants on stress-associated thymus involution

Male C57BL/6 mice were stressed by immobilization for 1, 2, 3, or 5 h per day for 14 days, with subsequent assessment of (a) thymic involution, (b) in vitro migration of stressed mice bone marrow cells toward thymocyte culture supernatants from neonates and from control or stressed mice, (c) composition of the bone marrow cell population, and (d) in vitro migration of normal bone marrow cells toward stressed mice thymocyte culture supernatants. The results obtained support the view that the reduced repopulation of thymus by precursor T cells contributes to thymus involution associated with stress. It is further shown that this effect could be owing to a reduction in the number of precursor T cells in the bone marrow, and/or to a diminished production of precursor T-cell chemoattractants.     

Influence of age on the proliferation and peripheralization of thymic T cells

Bone marrow cells obtained from B10.Thy-1.1 mice (H-2b, Thy-1.1) were injected directly into the thymus of C57BL/6 mice (H-2b,Thy 1.2) of various ages. Thymocyte precursors in the injected donor-bone marrow cells could proliferate in the thymic microenvironment in the following manner: first, preferentially proliferating into the subcapsular cortex; and second, spreading to the whole layer of the cortex, a portion of them gradually moving into the medulla. The proliferation of donor-type thymocytes was most pronounced when intrathymic injection of bone marrow cells (ITB) was performed in newborn mice and especially prominent in week-old mice; it took approximately ten weeks for donor-type thymocytes to finish the whole course of proliferation, differentiation, and emigration to the periphery. When ITB was performed in mice 4 weeks of age and older, the proliferation of donor-type thymocytes was retarded at onset, less pronounced in magnitude, and disappeared earlier. Emigration of donor-type T cells from the thymus to the peripheral lymphoid tissues occurred most rapidly when ITB was performed in newborn mice, and these T cells continued to reside thereafter in the peripheral lymphoid tissues. However, when ITB was performed in mice 4 weeks of age and older, the number of emigrated T cells in the spleen decreased (about a tenth of that in newborn mice) and, moreover, these T cells resided only transiently in the spleen. It was suggested that T cells emigrating from the thymus of mice from newborn to 2 weeks of age are long-lived, whereas those from the thymus in mice 4 weeks of age and older are short-lived. However, when 4-week-old young adult mice were treated by irradiation or hydrocortisone, the thymic capacity was enhanced in terms of proliferation and peripheralization of thymocytes, and emigrated T cells became long-lived.     

Modulation of B-cell abnormalities in lupus-prone (NZB x NZW)F1 mice by normal bone marrow-derived B-lineage cells.

(NZB x NZW)F1(NZB/WF1) mice spontaneously develop an autoimmune disease characterized by abnormality of haemopoietic stem cells. The present study examined a possible regulatory cell interaction between NZB/WF1 and normal bone marrow cells using radiation-induced chimeras. We demonstrated that the ability of NZB/WF1 bone marrow cells to transfer the typical disease with hypergammaglobulinemia including autoantibodies into lethally irradiated normal recipients was prevented by cotransfer of bone marrow from normal CBA/J mice but not from xid CBA/N mice carrying a selective defect in B-cell function. Flow cytometric analysis revealed that the generation of NZB/WF1 cells was reduced in the mixed chimeras given CBA/J but not CBA/N bone marrow cells. Interestingly, radiation chimeras reconstituted with a mixture of NZB/WF1 bone marrow and CBA/J splenic B cells did not show elevation of serum immunoglobulin levels, although most of the spleen cells were dominated by NZB/WF1 cells. On the other hand, NZB/WF1 B cells maturated in vivo in the presence of CBA/J bone marrow or splenic B cells lost the hyper-responsiveness to lipopolysaccharide (LPS) in the autoantibody production in vitro. These results suggest that radiosensitive normal B-lineage cells have the regulatory activity to ameliorate the hypergammaglobulinemia of NZB/WF1 mice by reducing the generation of NZB/WF1 B cells and/or by correcting their hyper-responsiveness, and that NZB/WF1 mice may have a defect(s) in the regulatory cell function. In addition, CBA/J splenic B cells were shown to modulate the B-cell abnormality even when injected into non-irradiated NZB/WF1 mice manifesting autoimmunity.     

Strain differences in the immune response of mice: II. Responses by neonatal cells in irradiated adult hosts

Lethally irradiated NZB and C57BL mice were injected with syngeneic thymus and marrow, or thymus and liver cells, and immunized with sheep red blood cells (SRBC). As in intact neonatal mice, NZB plaque-forming cell (PFC) responses were significantly higher than C57BL.

Irradiated (NZB×C57BL)F₁ and (NZB×BALB/c)F₁ hybrid mice were given mixtures of parental and syngeneic cells, and it was shown that the high NZB response to SRBC was characteristic of the newborn liver rather than of the thymus. NZB liver gave rise to more PFC against SRBC than BALB/c liver, but not more against chicken RBC. It is argued that liver cells contain genetic information regarding antibody specificity.

C57BL cells grew poorly in F₁ hosts, but the low SRBC response appeared to be characteristic of both liver and thymus.

The nomenclature of the participating cells, and their role in the development of immunological responsiveness, are discussed.

     

AGE-RELATED CHANGES OF THYMUS – MORPHOLOGICAL and FUNCTIONAL ASPECTS –

The thymus involutes progressively throughout life, beginning at around the sexual maturation. In long-lived BG3F₁ hybrid mice, the thymic capacity to induce T cell differentiation begins to decline earlier than the onset of thymic involution, although the magnitude of the decline is different by the subpopulation of T cells. Morphologically, the most active secretory structure seems to be limited exclusively to the neonatal thymus and certain structural changes, reflective of a decline in secretory function, can be detected early in life and they become more pronounced with age. Heterogeneity of thymic epithelial cells is suggested by the facts that age-related and radiation induced decline of immune activites are different in degree by subpopulation of T cells, and the concept is also supported from a morphological viewpoint. It is thus apparent that age-related thymic involution results in decrease of recruitment of fresh capable T cells and increase of old exhausted T cells, eventually bringing about T cell insufficiency in the aged individuals. Such an impaired immune function in the aged mice can be effectively restored by the combined grafting of young bone marrow and newborn thymus, and the thymus is apparently the most limiting factor in the aged. The biological significance of age-related thymic involution is also discussed.