Incomplete restoration of t-cell reactivity in nude mice after neonatal allogeneic thymus grafting

Grafting an allogeneic neonatal thymus to a congenitally athymic adult nude mouse allows a sufficient generation of φ-positive lymphocytes to bring their relative and absolute numbers close to the values found in normal mice. However, the T-cell characteristic reactivities of thymus-grafted nudes persistently remain low, especially the responsiveness of lymphoid cell suspensions to T-cell mitogens. In a series of thymus-grafted nudes, significant numbers of thymus donor type lymphocytes were found to persist for long periods of time within the grafted thymus, but their persistence could not be directly correlated with the acceptance of skin of same strain origin as the thymus or with responsiveness to mitogens.     

Sequential appearance of host-derived T cell subsets during differentiation in nude mice grafted with rat fetal thymus

To elucidate the abnormality of T cell differentiation in nude mice grafted with rat fetal thymus that develop multiple-organ-localized autoimmune diseases, we examined sequential appearance of T cell subsets and expression of TCR genes in BALB/c nude mice after grafting with fetal F344 rat thymus. We observed progressive expression of TCR gamma/delta-alpha/beta genes in the lymph node (LN) cells from 8 to 12 wk after grafting. An appreciable number of CD4+ T cells but few CD8+ T cells were detected in the LN at 8 wk after grafting. CD8+ T cells increased slowly in number by 12 wk after grafting but remained at a low level in comparison with those in nude mice 12 wk after grafting with BALB/c thymus. In correlation with an increase in the number of T cells expressing TCR alpha/beta genes, alloreactivity as assessed by MLR was increased to a normal level. However, CTL activity against alloantigens remained at a low level in the LN cells at 12 wk. At this stage, organ-specific autoimmune diseases and a high level of anti-DNA autoantibodies were detected. In these mice host-reactive T cells such as V beta 3- or V beta 11-bearing T cells were virtually eliminated in the peripheral mature T cell pool, whereas T cells maturing in the fetal rat thymus significantly proliferated in response to donor-rat stimulator cells. These results suggest that the development of the autoimmune diseases may be ascribed to an impaired maturation of CD8+ T cells but not to failure in clonal elimination of host-reactive T cells in nude mice grafted with rat thymus.     

Split tolerance in nude mice transplanted with 2'-deoxyguanosine-treated allogeneic thymus lobes

To elucidate the acquisition of self tolerance in the thymus, full-allogeneic thymic chimeras were constructed. Athymic C3H and BALB/c nude mice were reconstituted with the thymic lobes of BALB/c and B10.BR fetuses, respectively, that were organ cultured for 5 days in the presence of 2'-deoxyguanosine. T cells in these chimeras were tolerized to the host MHC in both MLR and CTL assays. In contrast, T cells in the chimeras exhibited split tolerance for the thymic MHC haplotype. CTL specific for class I MHC of the thymic haplotype were generated not only from the peripheral T cells of the chimeras but also from thymocytes re-populated in the engrafted thymic lobes. However, T cells in these chimeras responded poorly to the class II MHC of the thymic haplotype in a standard MLR assay. In a syngeneic MLR culture upon stimulation with enriched APC of the thymic haplotype, only 22 to 48% of the responses were mediated by CD4+ cells, and proliferations of CD4- cells were prominent. There were no haplotype-specific suppressor cells detected which would cause the unresponsiveness to the thymic class II MHC. These results indicated that the thymic lobes treated with 2'-deoxyguanosine were defective in the ability to induce the transplantation tolerance for the class I MHC expressed on the thymus, although the same thymic lobes were able to induce the transplantation tolerance for the thymic class II MHC.     

Can tolerant allogeneic cells restore nude mice?

BALB/c-nunu mice have been injected with allogeneic or tolerant allogeneic spleen or thymus cells and sheep red blood cells (SRBC). From day 3 to day 10 the mice were bled daily and the antiSRBC antibody was assayed by hemagglutination. No difference was found between recipients of normal allogeneic cells or tolerant allogeneic cells. Both showed a transient response but the response was maintained only in recipients of congenic cells.     

Clonal deletion of self-reactive T cells at the early stage of T cell development in thymus of radiation bone marrow chimeras

Sequential appearance of T cell subpopulations occurs in the thymocytes of irradiated C3H/He mice (H-2k, Mls-1b2a, Thy-1.2) after transplantation with bone marrow cells of AKR/J mice (H-2k, Mls-1a2b, Thy-1.1) (AKR----C3H chimeras). The donor-derived thymocytes of AKR----C3H chimeras on day 14 after bone marrow transplantation (BMT) contained a large number of blastlike CD4+CD8+ cells which represent relatively immature thymocytes, whereas those on day 21 after BMT consisted of small sized CD4+,CD8+ cells which represent a great part in normal thymocytes. To define the developmental stage at which clonal deletion of self-reactive T cells occurs in adult thymus, we followed the fate of V beta 6- or V beta 11-bearing T cells in the donor-derived thymocytes at the early stage of AKR----C3H chimeras. Mature thymocytes expressing high intensity of V beta 6 or V beta 11, which are involved in recognition of Mls-1a or MHC I-E gene products, respectively, were deleted from the donor-derived thymocytes on day 21. Immature thymocytes expressing low intensity of V beta 6 in CD3low thymocyte fraction decreased in proportion, whereas those expressing low intensity of V beta 11 rather increased in proportion in the donor-derived thymocytes of AKR----C3H chimeras from day 14 to day 21 after BMT. These results suggest that the clonal deletion of V beta 6-positive cells occurs just at the stage of immature CD3lowCD4+CD8+ cells, whereas the clonal deletion of V beta 11-positive cells may begin at the transitional stage from CD3lowCD4+CD8+ cells to CD3high single positive cells. Timing of negative selection of thymocytes may vary in distinct T cells capable of recognizing different self-Ag.     

Effects of thymus grafts in nude mice transplated with human malignant tumors

The effect of neonatal thymus grafts implanted in nude mice previously transplanted with three different human malignant tumors - an adenocarcinoma of the colon, a malignant melanoma and a Burkitt's lymphoma - were studied. In all immunologically reconstituted animals tumord were rejected. Tumor rejection stated 2-3 weeks after thymus implantation, and was completed after 30-6 weeks. Histological examination of lymphoid tissues showed a correlation between immunological reconstitution and tumor rejection. The rejection process showed a characteristic histologic picture with 3 phases - an early, an intermediate and a late phase - these were similar in the three tumor types examined. The possible mechanisms of reconstitution and tumor rejection are discussed.     

Extrathymic clonal deletion of self-reactive cells in athymic mice

The repertoire of T cells present in congenitally athymic mice was studied by flow cytometric analysis on populations of T cells expanded polyclonally in vitro. Athymic (BALB/c x C57BL/6)F1 mice have levels of potentially autoreactive V beta 3- and V beta 11-bearing T cells that are significantly higher than those of euthymic CB6F1 mice. Examination of potentially autoreactive cells in athymic AKR mice, however, yielded contrasting results. V beta 6+ cells, which are deleted intrathymically in normal AKR mice, are present in the repertoire of young (less than 6-wk-old) AKR nu/nu mice. Isolation of a cloned CD4+V beta 6+ cell line with Mls-1a reactivity from young AKR nu/nu mice indicates that the correlation between TCR usage and specificity is consistent with that described in euthymic mice and that this population contains autoreactive T cells that are not anergic. By 6 mo of age, however, cells expressing V beta 6 are no longer detectable. Inability to detect these cells is not simply caused by failure to expand these cells in culture, because freshly isolated populations from old nude mice exhibit the same selective absence of V beta 6-bearing cells. The data strongly suggest that extrathymic deletion, rather than clonal anergy, accounts for the apparent absence of autoreactive V beta 6-bearing cells in aged AKR nu/nu mice.     

Activation and control of self-reactive gammadelta T cells

Mammalian and avian CD3+ T cells can be separated into two lymphocyte subsets bearing heterodimeric T-cell receptors (TCR) composed of either alphabeta or gammadelta chains. Although it is now widely accepted that gammadelta and alphabeta T cells fulfill mandatory and nonredundant roles, the generality of this assumption and the exact functions played by gammadelta T cells remain uncertain. While an early protective role of gammadelta T cells has long been suspected, recent observations drawn in particular from transgenic models suggest their implication in the homeostatic control of immune and nonimmune processes. This hypothesis is also supported by the existence of several self-reactive gammadelta T-cell subsets in rodents and humans, whose specificity and effector properties will be detailed and discussed here. The present review will also describe several mechanisms that could allow efficient control of these self-reactive subsets while permitting expression of their regulatory and/or protective properties.     

A comparison of H-2 restriction of helper function using T cells from radiation chimeras and nude mice bearing thymus grafts

T cells from nude chimeras and radiation chimeras were tested for ability to cooperate with B cells of the thymus epithelium strain and B cells of the T-stem-cell strain in nude mice of appropriate strain backgrounds and also in irradiated F₁'s together with bone marrow cells of the two relevant strains (i.e., A-nu ← B or A → A × B chimera T cells were tested in A-nu and B-nu and in irradiated A × B F₁ with A bone marrow cells or B bone marrow cells). Whether the donor chimeras were antigen primed or not, all cells, when tested in nudes, provided help for the T-stem-cell type only. In contrast, in most cases, chimera T cells, tested in irradiated F₁'S, provided help for bone marrow cells of both thymus and T-stem-cell strains. These data indicate the involvement of two helper cell populations and an hypothesis to explain their interaction is presented.     

Suppressor T cells in thymus-reconstituted nude mice: regulation of mitogen-induced transformation

A population of glass-wool-adherent splenic cells which could suppress the response of other spleen cell populations to T-cell mitogens was isolated from thymus-reconstituted nude mice. Such adherent cells were characterized as sensitive to anti-Thy 1.2 and complement treatment. Glass-wool-adherent cells from athymic mice do not have suppressor activity to self or normal littermate NAC; however, these mice possess precursor suppressor cells as demonstrated by isolation of glass-wool-adherent T regulatory cells in thymus-grafted nude mice. Such cells are generated in either freshly obtained or in vitro cultured thymus. Evidence for suppressor T cells of host genotype was supported by their sensitivity to host-specific anti-Thy serum treatment as well as their generation in alymphoid thymus grafts. Prior anti-Thy 1.2 treatment of GAC partially removed the suppressor activity: however, macrophages and B lymphocytes were shown not to be secondary regulatory cells or suppressor mediators, thus mature T lymphocytes with low amounts of Thy 1.2 antigen may be responsible for this residual suppression. Further characterization of GAC indicates that active cell growth is required for their regulatory function, as irradiation removed the suppressor activity. This report provides evidence for the presence of a T-lymphocyte subpopulation which has a regulatory function and requires a thymus in the generation of these cells.     

Immune regulation by self-reactive T cells is antigen specific

Immune regulation plays an important role in the establishment and maintenance of self-tolerance. Nevertheless, it has been difficult to conclude whether regulation is Ag specific because studies have focused on polyclonal populations of regulatory T cells. We have used in this study a murine transgenic model that generates self-reactive, regulatory T cells of known Ag specificity to determine their capacity to suppress naive T cells specific for other Ags. We show that these regulatory cells can regulate the responses of naive T cells with the same TCR specificity, but do not inhibit T cell proliferation or differentiation of naive T cells specific for other Ags. These results demonstrate that immune regulation may be more Ag specific than previously proposed.     

Requirement of the thymus for the recovery of anti-alloantigen helper T cells from tolerance induced by intravenous presensitization with allogeneic cells

Intravenous presensitization of C57BL/6 (B6) mice with class I H-2-disparate B6-C-H-2bm1 (bm1) whole spleen cells and class II H-2-disparate B6-C-H-2bm12 (bm12) spleen cells depleted of APC resulted in almost complete elimination of the respective anti-bm1 and anti-bm12 reactivities. However, the reduced alloreactivities as assessed by Th cell capacities (proliferative responses and IL-2 production) were recovered around 8 wk after the i.v. presensitization in euthymic B6 mice. In contrast, background levels of bm1- or bm12-specific reactivities were revealed to last more than 12 wk after the presensitization in B6 mice which had been thymectomized prior to the i.v. presensitization. Such a reduced alloreactivity was also observed in the capacity to reject bm1 or bm12 skin grafts, and prolongation of graft survival was strikingly enhanced in the thymectomized group compared to that induced in the nonthymectomized group. However, there was an important difference in such prolongation in the thymectomized hosts between bm1 and bm12 grafts; a considerable percentage (greater than 80%) of bm12 skin grafts continued to take more than 5 mo, whereas about 90% of bm1 grafts were rejected by around 5 mo along with the emergence of weak, but detectable anti-bm1 Th and cytotoxic T cell activities. These results indicate that 1) i.v. presensitization regimen is capable of eliminating in vitro and in vivo alloreactive capabilities but these alloreactivities can be recovered in euthymic hosts with T cell repopulating potential and 2) there are differential requirements of the thymus for repopulating anti-bm1 (Lyt-2+) and anti-bm12 (L3T4+) T cell activities.     

Induction of self-reactive T cells after murine coronavirus infection.

We studied the mechanism of in vitro spontaneous lymphokine production by spleen cells from mice injected intraperitoneally with murine coronavirus stain JHM 1 month after infection, when infectious virus had already been cleared from the spleens. Removal of either CD4+ T cells or Ia+ antigen-presenting cells (APC) from the spleen cells abrogated interleukin-2 (IL-2) production. Addition of anti-CD4 or anti-Iad monoclonal antibodies to the culture suppressed IL-2 production. These results suggest that the response involved typical receptor-mediated activation of T cells. Surprisingly, reciprocal mixing experiments with a coculture of T cells from infected mice and APC from either infected or naive mice resulted in the production of IL-2. The absence of viral antigens in spleen cells 1 month after infection, as indicated by their inability to induce the proliferation of T-cell clones specific for the viral antigens, suggest that the T cells from mice 1 month after infection were not responding to the viral antigens. The inoculum components other than the virus did not induce this immune response. We also found that the frequency of self-reactive but not alloreactive IL-2-producing T cells in the spleens of infected mice was 3- to 10-fold higher than that in naive mice. These findings suggest that an increased frequency of self-reactive T cells which secrete IL-2 occurs following murine coronavirus infection. This may have important implications in the development of autoimmunelike phenomena following murine coronavirus infection.