Intrathymic Tolerance Induction: Determination of Tolerance to Class II Major Histocompatibility Complex Antigens in Maturing T Lymphocytes by a Bone Marrow-Derived Non-Lymphoid Thymus Cell

Two new experimental approaches were established to analyse the influence of the thymus on tolerance induction to major histocompatibility complex (MHC) antigens: The aim of the first experiment was to perform successful transplantation of adult allogeneic thymus tissue into nude mice, an attempt that has been unsuccessful in the past. Tolerance for the MHC genotype of a prospective thymus graft recipient (A) was induced in mice of strain B by injection of (A X B) splenocytes during the neonatal period. Adult thymic tissue obtained from these allogeneic donors (B) were grafted into the nude mice of strain A. The allogeneic thymus was accepted by the nude mice and immunoreconstitution was achieved. Subsequently the recipients developed tolerance to the MHC antigens of the allogeneic thymus donor as proved by mixed lymphocyte cultures and the acceptance of skin grafts. The second experiment was designed to determine which Ia-positive thymic compartment participates in conferring tolerance to MHC antigens in maturing T lymphocytes. Chimaeric thymus grafts were created by transplantation of neonatal thymus (A) into allogeneic nude mice (B) for a period of 8 weeks. The graft was populated with host bone marrow-derived Ia antigen-positive cells. The chimaeric thymuses consisting of type A epithelium but populated with both type A and B lymphocytes and non-lymphoid cells (i.e. Ia-positive macrophages and dendritic cells), were newly transplanted into nude mice of strain A. The engraftment lead to immunological reconstitution and the nude mice acquired tolerance to the MHC antigens expressed by the allogeneic Ia-positive cells populating the chimaeric graft. Irradiation of the chimaeric thymus prior to transplantation allowed transplantation of chimaeric thymus devoid of living thymocytes but still populated with functionally intact Ia-positive non-lymphoid cells. Transplantation of irradiated chimaeric thymuses resulted in immunoreconstitution and induced exactly the same allotolerance pattern as described above. The results demonstrate that not thymus epithelial cells but a bone-marrow-derived non-lymphoid thymus cell, most likely the Ia-antigen-positive thymic macrophage of dendritic cell, is responsible for the induction of tolerance to MHC antigens in developing T lymphocytes.     

T-cell tolerance induced in nude mice grafted with thymic epithelium.

This study investigated the ability of foetal thymic epithelium prepared by 24 degrees culture (24 degrees-TE) or treatment with deoxyguanosine (dGuo-TE), to induce tolerance in nude mice. Thymic chimeras were constructed in which the thymic epithelium differed from the host at both major histocompatibility complex (MHC) antigens and multiple minor histocompatibility antigens (mHa), or at mHa only. Peripheral CTL from nude mice receiving dGuo-TE disparate for mHa, or both MHC antigens and mHa, were uniformly tolerant of thymic mHa. CTL from nude mice grafted with 24 degrees-TE or dGuo-TE were tolerant of host MHC antigens, but the two treatments differed in the efficiency with which they induced tolerance to thymic MHC antigens. CTL responses specific for thymic MHC antigens could be generated in vitro from dGuo-TE grafted mice but not from those receiving 24 degrees-TE. The addition of concanavalin A (Con A) supernatant had no effect on the CTL tolerance observed in 24 degrees-TE grafted mice, suggesting that the lack of CTL responses was not due to tolerance in MHC class II restricted 'helper' cells. However, CTL responses against the thymic MHC antigens of dGuo-TE grafted mice displayed high sensitivity to blocking by anti-CD8 antibodies, indicating that these CTL were of low affinity. These results suggest that 24 degrees-TE induces tolerance in most thymic MHC-specific CTL precursors, whereas dGuo-TE induces tolerance only in CTL with high affinity for thymic MHC antigens. Therefore, 24 degrees-TE and dGuo-TE are both capable of inducing CTL tolerance, consistent with the previously reported acceptance of thymic donor-type skin grafts by nude recipients of dGuo-TE treatment. We conclude that MHC class I molecules on thymic epithelium play a role in negative selection of the developing T-cell repertoire.     

Thymocytes appear to ignore class I major histocompatibility complex antigens expressed on thymus epithelial cells

BALB/c nu/nu mice were grafted with embryonic 14-day-old C57BL/6 thymi which were transplanted either nontreated or after elimination of hemopoietic cells with 2-deoxyguanosine. In both types of grafts host cells developed normally into functional thymocytes. Thymocytes from 2-deoxyguanosine-treated but not from untreated grafts contained as many cytolytic T lymphocyte precursors specific for class I MHC antigens on thymus epithelium as normal BALB/c thymocytes. As cytolytic T lymphocyte precursors were neither suppressed nor activated in these grafts it is concluded that thymocytes ignore class I MHC antigens expressed on thymus epithelium.     

Induction of mouse syngeneic MLR by in vivo xenogeneic immunization with HLA-DR antigens

To study in mice the effects of in vivo xenogeneic immunization with human major histocompatibility complex (MHC) class II antigens, the animals were injected with HLA-DR antigens and their proliferative responses tested in vitro. The results showed that small amounts of HLA-DR proteins, acting as nominal antigens, were not only able to prime mice for a secondary in vitro xenogeneic mixed lymphocyte reaction but also induced a syngeneic mixed lymphocyte reaction. In contrast, allogeneic or syngeneic immunization of mice with soluble MHC class II molecules failed to stimulate an autoreactive response. The syngeneic mixed lymphocyte reaction was primarily directed against syngeneic MHC class II molecules since the murine T lymphocytes reacted against MHC class II-positive dendritic spleen cells and MHC class II-transfected mouse fibroblasts. A self-reactive T-cell line induced under these experimental conditions did not react in xenogeneic and allogeneic mixed lymphocyte reactions. However, these T lymphocytes proliferated when human peripheral blood lymphocytes of various haplotypes were presented in the context of syngeneic mouse antigen presenting cells.     

T cell-mediated cytotoxic immune responsiveness of chimeric mice bearing a thymus graft fully allogeneic to the graft of lymphoid stem cells

Fully allogeneic, chimeric mice were established by adult thymectomy of (A x B) F1 animals, grafting parental A-type thymus under the kidney capsula, followed by lethal (900 rd) irradiation and reconstitution with B parental-type bone marrow cells treated with xenogeneic anti-T cell antiserum plus complement. Following in vivo sensitization with inactivated Sendai virus (SV) suspensions, no virus-specific T cells could be detected within the spleen cells of the mice. Upon stimulation with third-party allogenic cells in a primary mixed lymphocyte culture, spleen cells of all animals generated alloreactive cytotoxic T lymphocytes (CLT). More interestingly, upon secondary in vitro stimulation with inactivated SV-conjugated B-type stimulator cells, B-type-restricted, virus-specific CTL were inducible in each case. Upon restimulation with SV-conjugated A-type stimulator cells, A being the H-2 type of the grafted thymus, T cells of some but no all mice generated A-type-restricted, virus-specific CTL. The data suggest that in allogeneic, chimeric mice virus-specific CTL can be induced. Moreover, virus-specific CTL, restricted to the H-2 type of the lymphoid stem cell inoculum, are more readily inducible than those restricted to the H-2 type of the allogeneic thymus.     

The myelin basic protein-specific T cell repertoire in (transgenic) Lewis rat/SCID mouse chimeras: preferential Vβ8.2 T cell receptor usage depends on an intact Lewis thymic microenvironment

In the Lewis rat, myelin basic protein (MBP)-specific, encephalitogenic T cells preferentially recognize sequence 68–88, and use the Vβ8.2 gene to encode their T cell receptors. To analyze the structural prerequisites for the development of the MBP-specific T cell repertoire, we reconstituted severe-combined immunodeficient (SCID) mice with fetal (embryonic day 15–16) Lewis rat lymphoid tissue, and then isolated MBP-specific T cell lines from the adult chimeras after immunization. Two types of chimera were constructed: SCID mice reconstituted with rat fetal liver cells only, allowing T cell maturation within a chimeric SCID thymus consisting of mouse thymic epithelium and rat interdigitating dendritic cells, and SCID mice reconstituted with rat fetal liver cells and rat fetal thymus grafts, allowing T cell maturation within the chimeric SCID and the intact Lewis rat thymic microenvironment. Without exception, the T cell lines isolated from MBP-immunized SCID chimeras were restricted by MHC class II of the Lewis rat (RT1.B¹), and none by I-A^d of the SCID mouse. Most of the T cell lines recognized the immunodominant MBP epitope 68–88. In striking contrast to intact Lewis rats, in SCID mice reconstituted by rat fetal liver only, MBP-specific T cell clones used a seemingly random repertoire of Vβ genes without a bias for Vβ8.2. In chimeras containing fetal Lewis liver plus fetal thymus grafted under the kidney capsule, however, dominant utilization of Vβ8.2 was restored. The migration of liver-derived stem cells through rat thymus grafts was documented by combining fetal tissues from wild-type and transgenic Lewis rats. The results confirm that the recognition of the immunodominant epitope 68–88 by MBP-specific encephalitogenic T cells is a genetically determined feature of the Lewis rat T cell repertoire. They further suggest that the formation of the repertoire requires T cell differentiation in a syngeneic thymic microenvironment.     

Spontaneous in vivo retrovirus-infected T and B cells, but not dendritic cells, mediate antigen-specific Fas ligand/Fas-dependent apoptosis of anti-retroviral CTL

C57BL/6 (H-2b), but not spontaneous virus-expressing AKR.H-2b congenic, mice generate retrovirus-specific CD8+ CTL responses to the immunodominant Kb-restricted epitope, KSPWFTTL. AKR.H-2b non-responsiveness is mediated by a peripheral tolerance mechanism. When co-cultured with primed B6 antiviral pCTL, AKR.H-2b splenocytes are recognized by the antiviral TcR as "veto" cells, which inhibit by an exquisitely virus-specific, MHC-restricted, veto cell FasL/responder T cell Fas, mediated apoptotic mechanism. Here, AKR.H-2b thymus, lymph node, and bone marrow cells are also shown to inhibit antiviral CTL generation. Purified AKR.H-2b CD4+ and CD8+ T cells, and B cells, served effectively as FasL-dependent veto cells. In contrast, AKR.H-2b dendritic cells (DC) did not efficiently veto antiviral CTL responses, despite expressing sufficient MHC class I/viral peptide complexes for TcR recognition. AKR.H-2b DC also expressed FasL mRNA and cell surface protein, albeit at a lower level than AKR.H-2b T and B cells. These findings suggest a fail-safe escape mechanism by virus-infected cells for escape from CTL-mediated immunity.     

Serological analysis of H-2 antigens expression on the cell surface of selected mouse tumors

Studies on histocompatibility antigens expression on different selected mouse tumor cells revealed very variable pattern. No qualitative alterations in H-2 antigens expression on the cells of the following tumors: RL male 1 and MP26a of BALB/c (H-2d) mice, ASL-1 and RADA-1 of A (H-2a) mice, EL-4 and E male G2 of C57B1/6 (H-2b) mice, K36 and AKSL-4 of AKR (H-2k) mice could be detected. However, the quantitative differences in H-2 antigens expression on some of these tumors were observed.     

IgG antibody and delayed-type hypersensitivity responses in nude mice grafted with thymic epithelium.

This study compared the ability of foetal thymic epithelium depleted of lymphocytes and dendritic cells, by low temperature or deoxyguanosine (dGuo) treatment in organ culture, to reconstitute T-cell function in nude mice. It is shown that renal capsule grafts of either type could promote the development of functional T lymphocytes in the periphery, as judged by in vivo assays. Both syngeneic and allogeneic thymic epithelium endowed nude mice with the capacity to mount IgG antibody and delayed-type hypersensitivity (DTH) responses to the T-dependent antigen ovalbumin (OVA). Functional reconstitution was accompanied by the appearance of Thy-1-bearing cells in the spleens of thymic grafted nude mice. The results from allogeneically grafted recipients show that a substantial population of peripheral T cells was present that collaborated with B cells and other antigen-presenting cells (APC) which do not express major histocompatibility complex (MHC) molecules of the thymus donor haplotype.     

Achievement of cellular immunity and discordant xenogeneic tolerance in mice by porcine thymus grafts

Specific cellular immune tolerance may be essential for successful xenotransplantation in humans. Thymectomized (ATX), T and NK ceil-depleted immunocompetent mice grafted with xenogeneic fetal pig thymic and liver tissue (FP THY/LIV) result in efficient mouse thymopoiesis and peripheral repopulation of functional mouse CD4^ T cell.Very importantly, the reconstituted mouse T cells are specifically tolerant to pig donor antigens. Studies demonstrated that porcine MHCs mediated positive and negative selection of mouse thymocytes in FP THY grafts, whereas mouse MHCs were involved in negative selection in grafts. Therefore, T cell tolerance to xenogeneic donor antigens could be induced by grafting donor thymus tissue. Xenogeneic thymic replacement might have a potential role in the reconstitution of cellular immunity in patients with AIDS or other immunodeficiencies caused bv thvmus dvsfunction.     

Autoimmune diseases developed in athymic nude mice grafted with embryonic thymus of xenogeneic origin.

Reconstitution of T cell functions in athymic BALB / c nude mice was investigated after transplantation of embryonic thymus grafts under the renal capsule (TG nude mice). Thymi were obtained from mice, rats, rabbits, hamsters, guinea pigs, swine and cows. All of the grafted thymi grew and formed proper thymic structures with host CD90(+) cells. The TCRalpha beta expression pattern of the lymphocytes in the grafted thymi was quite similar to that of normal mouse thymocytes. Sufficient CD4(+) populations were observed in the peripheral lymphoid organs of all groups of TG nude mice. Plaque-forming cell assay revealed that the TG nude mice had acquired considerable helper T cell functions. Histological and serological studies, however, showed multiple organ-localized inflammatory diseases in TG nude mice of all xenogeneic thymus groups, but not with syngeneic or allogeneic thymi. The organs affected were the thyroid, the lacrimal, submandibular and sublingual glands, the eyes, stomach and ovaries. The incidence of lesions was variable depending on the species of origin of the grafted thymus. Lesions from TG nude mice were successfully transferred into naive nude mice by host CD4(+) cells. These results together suggested that the microenvironment of grafted thymi, even if xenogeneic, is able to educate host T cell precursors. However, this reconstitution of functions does not always induce tolerance to certain autoantigens, resulting in development of multiple autoimmune lesions.     

The interaction defect of accessory molecules is responsible for the poor ex vivo response to human antigens of mouse T helper cells

Mouse T cells fail to respond to xenogeneic pig and human antigens using the direct antigen‐presenting pathway. The poor response by mouse CD8 cells is because of multiple defects in the molecular interactions between mouse CD8 cells and xenogeneic antigen‐presenting cells (APCs). Using human CD4/DR3⁺, mouse CD4^?/major histocompatibility complex (MHC) class II ^? mice, we investigated the defects in molecular interaction responsible for the poor response to xenogeneic antigens by naïve mouse CD4⁺ cells. Mouse CD4 cells failed to respond to human leucocyte antigen (HLA)‐DR3 expressed on mouse APCs but developed a strong response to alloantigens, indicating a defect in the interaction between mouse CD4 and HLA‐DR3 molecules. Human CD4⁺/mouse CD4^– mouse T cells respond poorly to human and pig APCs but not allogeneic APCs, indicating that accessory molecular interactions are deficient across highly separated species. Adding mouse interleukin‐2 (IL‐2) to the mixed lymphocyte reaction system did not improve the poor response to human or pig antigens by mouse or human CD4⁺/mouse CD4^– mouse T cells. Therefore, multiple molecular interactions deficient between mouse CD4 cells and human or pig APCs may lead to the poor response to xenogeneic antigens by naïve mouse CD4 cells.     

Development of T cells in SCID mice grafted with fetal thymus from AKR mice or F344 rats

To examine the development of T cells within an allogeneic or xenogeneic environment, we engrafted the fetal thymus from AKR mice or F344 rats under the kidney capsule of SCID mice (mTG and rTG mice). T lymphopoiesis developed in SCID mice 2 months after transplantation, although the ratio of CD4/CD8 in both experimental groups was different from that of normal control. T cells in mTG mice did not show in vitro proliferation or cytotoxicity against either host-type C.B-17 (H-2^d) or donor-type AKR (H-2^k) cells, while they exerted potent activities against third-party BIO (H-2^b) cells. In contrast, T cells in rTG mice exhibited proliferation against both host-type C.B-17 and donor-type F344 rat cells. Consistently, graft-vs.-host disease symptoms developed in these mice and histological examination showed impressive infiltration of lymphocytes into the skin or into the mucosal layers of the stomach. Activated state of T cells in rTG mice was also evidenced by the positive expression of interleukin-2 receptor. Taken together, fetal thymus appears to contain progenitor cells which are sufficient for in vivo reconstitution of T lymphopoiesis, but species-specific environment is important for the induction of tolerance. In mTG mice, Vβ6⁺ T cells reactive to donor Mls^a determinants and Vβ3⁺ T cells reactive to host Mls^c determinants were deleted, suggesting that tolerance was regulated mainly by clonal deletion. By contrast, Vβ11⁺ T cells reactive to Mls^f determinants were not deleted possibly due to the lack of their ligands.     

Transplantation of T cell-mediated, lymphoreticular disease from the scurfy (sf) mouse.

The X-linked mutation, scurfy (sf), causes a fatal lymphoreticular disease characterized by runting, lymphadenopathy, splenomegaly, hypergammaglobulinemia, exfoliative dermatitis, Coombs'-positive anemia, and death by 24 days of age. T lymphocytes are required to mediate this syndrome as shown by a total absence of disease in mice bred to be scurfy and nude (sf/Y; nu/nu). The scurfy phenotype is not transmitted by sf/Y bone marrow transplants, though cells of scurfy origin do reconstitute all lymphoid organs in the recipient mouse. These data suggest that scurfy disease results from an abnormal T cell development process and not from an intrinsic stem cell defect. We therefore tested the ability of transplanted scurfy thymuses to transmit scurfy disease to congenic euthymic mice, to athymic (nude) mice, and to severe combined immunodeficiency (SCID) mice. Euthymic recipients of sf/Y thymic grafts remained clinically normal as did all SCID and nude recipients of normal thymus transplants. Morphological lesions similar to those found in scurfy mice occurred in all H-2-compatible nude and SCID recipients of sf/Y thymic grafts. Intraperitoneal injections of scurfy thymocytes, splenocytes, and lymph node cells also transmitted the scurfy phenotype to H-2-compatible nude mice and SCID mice. Our findings indicate that scurfy disease can be transmitted to T cell-deficient mice by engraftment of scurfy T cells, but that pathogenic scurfy T cell activities can be inhibited (or prevented) in immunocompetent recipient mice.     

Differentiation antigens in rat lymphocyte subpopulations recognized by rabbit antisera against electrophoretically separated cells.

Rat lymphocytes of high (H cells) and low (L cells) electrophoretic mobility (EPM) from lymph node (LN), spleen (Spl) and thymus (Thy) were separated by free flow electrophoresis and antisera against these cells raised in rabbits. Eight distinct lymphocyte-specific xenoantigens could be characterized by appropriate cross-absorption of three antisera: anti-Thy-L cell IgG, anti-LN/-Spl-H cell IgG and anti-LN/Spl-L cell IgG. Cross-absorption of anti-Thy-L cell IgG with unseparated LN or Spl cells revealed a thymus-specific antigen complex which was absent from peripheral rat lymphocytes but present on mouse thymocytes and peripheral T cells. This complex could be further separated into brain-associated and nonassociated antigens. The brain-associated antigens consisted of a rat-specific component, a component shared by the rat and all mice which carry the Thy-1.1 and Thy-1.2 allogenic specificity, and a third component shared by rat and Thy-1.1 mice. This Thy-1 antigen complex showed a 26-fold larger antibody-binding capacity to thymocytes of low EPM than to those of high EPM. The thymic antigens which were not absorbed by brain tissue showed similar antibody-binding capacity to all thymocytes whether of low or high EPM. Anti-LN/Spl-H cell IgG cross-absorbed with LN/Spl-L cells detected antigens which were localized on all rat peripheral H cells and thymocytes but which were absent from peripheral L cells. Further absorption with Thy-L cells revealed a distinct subclass of antigens present on peripheral H cells and Thy-H cells but absent from Thy-L cells. On the other hand, cross-absorption of anti-LN/Spl-L cell IgG with peripheral H cells provided specificities which were directed against all peripheral L cells but which did not react with peripheral H cells and thymocytes. The relationship of these different xenoantigens to known lymphocyte subpopulations is discussed.     

Aberrant MHC antigens in a sarcoma virus-induced mouse tumour

From a series of mouse sarcomata, newly induced by Rous sarcoma virus (RSV), the DOH cell line was shown to lack expression of syngeneic H-2Kd and Dk antigens (Noll et al., 1986). It unexpectedly displayed determinants specific for H-2Kk molecules. Interferon treatment of DOH stimulated the expression of H-2Kk determinants and also the display of some, but not all, determinants of the syngeneic H-2Kd molecules. H-2Dk expression was not stimulated. Southern blot hybridization of genomic DNA digests from DOH cells confirmed the identity of the H-2K region with that from syngeneic C3H.OH liver cells, but also showed changes in the pattern of restriction fragments that contain class I genes from the D and Qa regions. These results suggest that aberrant MHC class I molecules that carry H-2Kd- and H-2Kk-like determinants are expressed by DOH sarcoma cells. These molecules may act as target antigens for tumour-specific cytotoxic T cells elicited by injection of DOH cells into syngeneic mice.     

Rat stem cells developing in irradiated SCID mice fail to become tolerized and cause lethal graft-versus-host disease.

Graft-versus-host disease (GVHD) is prominent in irradiated hosts given whole allogeneic bone marrow cells but is generally undetectable when T-depleted stem cells are transferred; under these conditions, the mature T cells arising from the donor stem cells become tolerant to host antigens and fall to cause GVHD. We show here that a radically different situation can occur when hosts are reconstituted with xenogeneic stem cells. When lightly irradiated, adult C.B-17 SCID mice injected with Lewis rat fetal liver (FL) cells show near-total repopulation with rat-derived lymphohemopoietic cells, including T and B cells. However, in marked contrast to chimeras prepared with allogeneic mouse FL cells, rat FL-->SCID chimeras develop severe and often lethal chronic GVHD. In these rat-->mouse chimeras, the rat T cells show limited tolerance to host mouse antigens as determined by various parameters including mixed lymphocyte reaction and cytotoxic T lymphocyte assays in vitro, adoptive transfer of T cells to secondary SCID hosts, and the lack of V beta deletion to endogenous host mtv antigens. GVHD in irradiated rat-->SCID chimeras is most prominent with Lewis FL but also applies to Fisher 344 and Wistar Furth FL cells. The failure of newly formed rat T cells in rat-->SCID chimeras to become fully tolerant to host mouse antigens appears to be due to depletion of host antigen-presenting cells by irradiation. Thus, rat-->SCID chimeras generated by transplanting rat FL cells into unirradiated neonatal SCID mice fail to develop GVHD, and the rat T cells display self-tolerance. As allogeneic H-2-different mouse FL-->irradiated SCID chimeras display strong self-tolerance, presumably through recognition of host antigens on thymic epithelial cells, the implication is that mouse thymic epithelial cells are tolerogenic only for mouse and not for rat immature T cells.     

Tolerogenicity of thymic epithelium

To attempt to resolve the controversy on the role of thymic epithelial cells (TEC) in tolerance induction, athymic mice were grafted with allogeneic day-14 fetal thymuses treated with deoxyguanosine in vitro. The data indicate that the tolerogenicity of TEC varies considerably according to the antigen and the subpopulation of T cells studied. For cytotoxic CD8+ cells responding to H-2 class I antigens, TEC induce minimal tolerance. For proliferative responses of CD4+ cells, by contrast, TEC induce significant tolerance to H-2 class II antigens but no detectable tolerance to Mlsa antigens.