Self-reactive T cell clones in a restricted population of interleukin-2 receptor β+ cells expressing intermediate levels of the T cell receptor in the liver and other immune organs

T cells expressing high levels of the T cell receptor (TCR^high) differentiate in the major intrathymic pathway and then distribute to the peripheral immune organs, whereas T cells expressing intermediate levels of the TCR (TCR^int) differentiate in both extrathymic pathways and an alternative intrathymic pathway and localize in unique sites, including the liver and thymic medulla. Since TCR^int cells constitutively express interleukin-2 receptor β-chain (IL-2R), two-color staining for CD3 (or TCR) and IL-2Rβ clearly distinguished IL-2RßT⁺ CD3^int (or TCR^int) cells from IL-2Rβ^?, CD3^high cells. CD3^int cells may be considered to be primordial T cells based on their phenotype, morphology and other functional properties. In this study, using anti-Vβ mAb in conjunction with the endogenous superantigen Mls, the distribution of self-reactive clones among T cells generated in all of the above pathways was investigated in mice. Self-reactive T cell clones were confined to IL-2Rβ⁺, CD^int cells, in all of the organs tested. A significant proportion of self-reactive clones was never identified among CD3^high cells in the thymus and peripheral immune organs in either young (8 week old) or old (50 week old) mice. Possibly reflecting their self-reactivity, CD3^int cells, but neither NK cells nor CD3^high cells had a potent cytotoxic effect against a syngeneic hepatoma in the presence of anti-CD3 mAb. These results raise the possibility that CD3^int cells seen in the liver and thymus might belong to a similar primordial lineage of T cells, and that self-reactive clones are not generated through the major intrathymic pathway, but only through extrathymic pathways and an alternative intrathymic pathway.     

Reduced development of CD4−8−B220+ T cells but normal autoantibody production in lpr/lpr mice lacking major histocompatibility complex class I molecules

The lpr gene has recently been shown to encode a functional mutation in the Fas receptor, a molecule involved in transducing apoptotic signals. Mice homozygous for the lpr gene develop an autoimmune syndrome accompanied by massive accumulation of double-negative (DN) CD4^?8^?B220⁺ T cell receptor-α/β⁺ cells. In order to investigate the origin of these DN T cells, we derived lpr/lpr mice lacking major histocompatibility complex (MHC) class I molecules by intercrossing them with β2-microglobulin (β2m)-deficient mice. Interestingly, these lprβ2m–/– mice develop 13-fold fewer DN T cells in lymph nodes as compared to lpr/lpr wild-type (lprWT) mice. Analysis of anti-DNA antibodies and rheumatoid factor in serum demonstrates that lprβ2m–/– mice produce comparable levels of autoantibodies to lprWT mice. Collectively our data indicate that MHC class I molecules control the development of DN T cells but not autoantibody production in Ipr/lpr mice and support the hypothesis that the majority of DN T cells may be derived from cells of the CD8 lineage.     

Phenotypic and functional modulation of T cells in vivo by extrathymic T cells when T cells with MHC class II disparity were injected into athymic nude mice

TCR^high cells are generated by the mainstream of T cell differentiation in the thymus, whereas TCR^int cells (or NK1.1⁺ T cells) are generated extrathymically in the liver and by an alternative intrathymic pathway. It is still unknown how these T cell populations interact in vivo with each other. To investigate the interaction of TCR^int cells with TCR^high cells, we used congenitally athymic nude (B6-nu/nu) mice which carry only TCR^int cells in all immune organs. When TCR^high cells from B6-C-H-2^bm12 (bm12) mice (i.e. I-A^bm12) were injected into B6-nu/nu mice (i.e. 1-A^b), the expanding T cell population was a mixture of TCR^high cells of donor origin and TCR^int cells of recipient origin. However, 9 Gy-irradiated nude mice permitted a full expansion of TCR^high cells which expressed the IL-2Rα⁺β⁺ phenotype, namely, they were at the most activated state. These mice died of acute graft-versus-host disease (GVHD) within 5 days. On the other hand, non-irradiated nude mice suppressed the expansion of TCR^high cells of donor origin and such TCR^high cells continued to have the IL-2Rα^±β⁺ phenotype. These mice could survive but showed signs of chronic GVHD thereafter. In both situations, CD4⁺αβ T cells expanded irrespective of donor or recipient origin. These results suggest that TCR^int cells in the recipient mice possess a regulatory function in relation to donor TCR^high cells; as a result, fully activated TCR^high cells acquired the IL-2Rα⁺β⁺ phenotype and injured the host, but TCR^high cells suppressed in vivo remained as the IL-2Rα^±β⁺ phenotype and only partially injured the host.     

Absolute dependence of T cell receptorhi cell generation and relative dependence of T cell receptorint cell generation on the thymus

Recent evidence indicates that conventional T cells are generated by the mainstream of T cell differentiation in the thymus and acquire a high density of T cell receptor expression (i.e. TCR^hi). In contrast, primordial T cells (or NK1.1⁺ T cells) are generated by the extrathymic pathways or an alternative intrathymic pathway and express an intermediate density of TCR (i.e. TCR^int). To obtain further evidence, it was examined how thymus grafting influenced the distribution of T cell populations in athymic nude mice. When BALB/c nu/nu mice were engrafted with thymocyte-depleted BALB/c+/+ fetal thymi, two changes emerged after grafting: nude mice generated TCR^hi cells de novo in the periphery as well as in the grafted thymi, and the absolute number of interleukin-2 receptor β chain⁺ TCR^int cells increased prominently in number in the periphery. Among thymic hormones tested, the administration of thymosin α induced a slight expansion of CD3^int cells in nude mice. To examine a possible interaction of TCR^int cells with TCR^hi cells in the periphery, B6 nu/nu mice (Ly5.2⁺) were injected with TCR^hi cells purified from the spleen of B6 Ly5.1 congenic mice. In this case, TCR^int (Ly5.2⁺) cells expanded well in all tested organs of nude mice. These results suggest that the generation of TCR^hi cells is absolutely dependent on the thymus and that TCR^int cells expand under the influence of the thymus (humoral) and due to interaction with thymus-derived conventional T cells.     

Restricted appearance of self-reactive clones into T cell receptor intermediate cells in neonatally thymectomized mice with autoimmune disease

Neonatally thymectomized (NTx) mice fall victim to such autoimmune diseases as gastritis and pancreatitis with aging. Self-reactive T cell clones are known to be consistently generated through TCR intermediate (i.e. TCR^int) cell differentiation in normal mice (i.e. via the extrathymic pathways and an alternative intrathymic pathway). It was investigated whether the generation of such clones in NTx mice follows this rule or whether they are generated by default via mainstream T cell differentiation in the thymus. The majority of T cells generated in NTx mice were TCR^int cells in all organs tested. In contrast to athymic mice, which carry only TCR^int cells with aging, a leaky appearance of high TCR (i.e. TCR^hi) cells emerged in the lymph nodes and other organs of NTx mice. Self-reactive clones estimated by anti-Vβ monoclonal antibodies in conjunction with the Mls system were confined to TCR^int cells in all tested organs, including a target organ, the stomach, in NTx mice. A leaky population of TCR^hi cells did not contain a significant number of self-reactive clones. Moreover, such self-reactive clones among TCR^int cells in NTx mice with autoimmune disease were shown to be nonanergic in the proliferation assay. The present results suggest that the generation of self-reactive clones is totally due to TCR^int cell differentiation, although it is still undetermined whether the expanding TCR^int cell population is generated via the extrathymic pathway or an alternative intrathymic pathway. It is shown here not to be due to a failure of the TCR^hi cell-differentiation pathway in NTx mice.     

Supportive cellular elements for hepatic T cell differentiation: T cells expressing intermediate levels of the T cell receptor are cytotoxic against syngeneic hepatoma,and are lost after hepatocyte damage

Extrathymic T cells exist in the liver and are often seen in close contact with Kupffer cells in the hepatic sinusoids. Since selective depletion of Kupffer cells has become possible by using liposome-encapsulated clodronate, it was investigated whether elimination of Kupffer cells influences the level of extrathymic T cells in the liver. Extrathymic T cells were identified as interleukin-2 receptor β-chain (IL-2Rβ) intermediate TCR (TCR^int) cells by two-color staining for CD3 or T cell receptor (TCR) and IL-2Rβ. The elimination of Kupffer cells did not significantly affect levels of TCR^int cells up to 7 days after treatment. We then examined monocyte colony stimulating factor (M-CSF)-deficient op/op mice (low levels of Kupffer cells). Extrathymic T cells both in the liver and spleen of these mice were detected at a level comparable to that of control mice. Since extrathymic T cells in the liver are sometimes located in the parenchymal space, the relationship between extrathymic T cells and hepatocytes was then examined. Electron microscopy revealed that some hepatic T cells adhered directly to hepatocytes. When hepatocytes were damaged by a single injection of CCl₄, hepatocyte death and subsequent hepatic fibrosis were induced. Beginning 3 days after injection, CD3^int cells, but not other type of cells, decreased prominently. Purified CD3^int cells, as well as whole lymphocytes in the liver, were cytotoxic against syngeneic hepatoma. In parallel with the above-mentioned hepatic damage, the cytotoxic activity of lymphocytes against such targets was impaired in the liver. These results suggest that extrathymic generation of TCR^int cells and their acquisition of cytotoxic function are relatively independent of Kupffer cells, but are dependent on hepatocytes.     

Lack of association of Vβ8+ T cells with lupus-like syndrome in MRL-lpr/lpr mice

To evaluate the role of Vβ8⁺ T cells in the development of lupus-like autoimmune syndrome in MRL-lpr/lpr mice, we treated them with the F23.1 anti-Vβ8 monoclonal antibody (mAb) from birth to 4 months of age. Here we report that almost complete depletion of Vβ8⁺ T cells by the F23.1 mAb treatment neither inhibited nor delayed the development of hypergammaglobulinemia, autoantibody production and autoimmune glomerulonephritis in MRL-lpr/lpr mice. In addition, the F23.1 mAb treatment did not prevent the development of lymphadenopathy and the generation of a CD4^?CD8^? double-negative T cell subset, characteristically accumulating in lpr lymph nodes. Our results strongly argue against the idea that the Vβ8⁺ T cells play a critical role in the development of lupus-like autoimmune syndrome in MRL-lpr/lpr mice.     

Elucidation of the protein kinase C-dependent apoptosis pathway in distinct subsets of T lymphocytes in MRL-lpr/lpr mice

MRL-lpr mice are severely impaired in the Fas pathway of apoptosis induction. We here evaluate another pathway of apoptosis induction in MRL-lpr mice which is protein kinase C (PKC) dependent. Despite the defect of the Fas pathway, apoptosis developed during culture in vitro in splenic T lymphocytes from MRL-lpr mice more extensively than in T lymphocytes from MRL-+/+ mice. Apoptosis induction in the former cells was then found to be greatly promoted by PKC inhibitor H-7, and partially prevented by PKC activator phorbol 12-myristate 13-acetate (PMA). High sensitivity to H-7, but not to PKA inhibitor HA 1004, of these cells for apoptosis induction was confirmed by detailed time course and dose-dependency experiments of the drug effect. Population analysis showed that both CD4⁺ T lymphocytes and CD8⁺ T lymphocytes from MRL-lpr mice were highly sensitive to H-7, whereas CD8⁺ T lymphocytes, but not CD4⁺ T lymphocytes, from MRL-+/+ mice were susceptible to the reagent. Interestingly, B220⁺Thy-1⁺CD4^?CD8^? T lymphocytes from MRL-lpr mice were most sensitive to H-7 for apoptosis induction. Correspondingly, the membrane-translocated activated PKC-α level in splenic T lymphocytes from MRL-lpr was more extensively up-regulated by PMA than in splenic T lymphocytes from MRL-+/+. These results suggest that some signal consistently activates PKC in MRL-lpr T lymphocytes, and this event is needed for survival of these cells. On the other hand, CD4⁺CD8⁺ thymocytes were deleted by apoptosis in culture with PMA, whether these thymocytes were from MRL-lpr mice or MRL-+/+ mice. This finding suggested that the apoptosis induction pathway linked to PKC activation is intact in CD4⁺ CD8⁺ thymocytes from the Fas-defective MRL-lpr mice. We conclude from these results that the PKC-dependent signal pathways for either cell death or cell activation are intact or even accelerated in lpr mice, which could both compensate for the loss of the Fas pathway and promote the generation of autoreactive T lymphocytes.     

Identification and cellular distribution of the rat interleukin-2 receptor β chain: induction of the IL-2Rα−β+ phenotype by major histocompatibility complex class I recognition during T cell development in vivo and by T cell receptor stimulation of CD4+8+ immature thymocytes in vitro

A mouse monoclonal antibody (mAb) to the rat interleukin-2 receptor β (IL-2Rβ) chain was generated using IL-2Rβ cDNA-transfected mouse L929 cells for immunization and differential screening. This antibody, called L316, detects a cell surface protein with an apparent molecular mass of about 80 kDa. In peripheral lymphoid organs of young adult rats, IL-2Rβ expression is restricted to T and natural killer (NK) cells, and less than 10% of IL-2Rβ⁺ cells co-express the IL-2Rα chain. IL-2Rβ was detected on all NKRP-1^hi (NK⁻) and NKRP-1^lo cells (T-lineage cells of unknown function), most peripheral γδ T cells and on 30–40% of CD8 and 10% of CD4 αβ T cells. In the adult rat thymus, mAb L316 detects a small subset (about 1%) of predominantly IL-2Rα⁻ cells which express cell surface markers characteristic of mature T lymphocytes and contain a high proportion of CD4⁻8⁻ and CD4⁻8⁺ αβ T cell receptor (TCR)⁺ thymocytes. TCR-V usage suggests that major histocompatibility complex (MHC) class I plays a more important role than MHC class II in the selection of these cells. On immature CD4⁺8⁺ rat thymocytes, IL-2Rβ cell surface expression is readily induced by TCR stimulation in vitro, supporting the idea that in vivo, the IL-2Rβ⁺ phenotype is the result of TCR engagement during thymic selection.     

CD5− CD8αβ intestinal intraepithelial lymphocytes (IEL) are induced to express CD5 upon antigen-specific activation: CD5− and CD5+ CD8αβ IEL do not represent separate T cell lineages

We followed αβ T cell receptor (TCR) usage in subsets of gut intraepithelial lymphocytes (IEL) in major histocompatibility complex class I-restricted αβ TCR-transgenic (tg) mice. The proportion of tg αβ TCR⁺ CD8αβ IEL is reduced compared with CD8⁺ splenocytes of the same animal, particularly under conventional conditions of maintenance. Further fractionation of CD8αβ IEL according to the expression level of surface CD5 revealed that in conventionally housed animals tg TCR⁺ CD5^? CD8αβ IEL are as frequent as in specific pathogen-free (SPF) mice, whereas tg TCR⁺ CD5^int or, even more pronounced, tg TCR⁺ CD5^hi CD8αβ IEL are greatly diminished when compared with mice kept under SPF conditions. Upon antigen-specific stimulation of CD5^? CD8αβ IEL in vitro, CD5 surface expression is up-regulated on a large fraction of cells within 48 h. Up-regulation of CD5 surface expression is further enhanced by the presence of the anti-αIEL monoclonal antibody 2E7. This clearly demonstrates that CD5^?, and CD5⁺ CD8αβ IEL cannot be considered as separate T cell lineages.     

A specific intercellular pathway of apoptotic cell death is defective in the mature peripheral T cells of autoimmune lpr and gld mice

Homozygosity for either of the unlinked murine autosomal recessive mutations lpr or gld leads to autoimmunity characterized by peripheral accumulation of CD4^?/CD8^? “double-negative” T cells, autoantibodies and various forms of tissue pathology. Recently, the gene affected by lpr was identified as fas, whose product acts as a trigger for programmed cell death or apoptosis. Data reported here indicate that the Fas receptor and its ligand, the wild-type form of the gld gene product, are essential for antigen-stimulated peripheral T cell apoptosis. Furthermore, the wild-type gld gene product is a non-cell-autonomous protein that is produced by activated T cells. Apoptotic elimination of antigen-receptor-triggered peripheral T cells appears to be abnormal in lpr and gld mice, and this deficiency causes peripheral T cells to accumulate resulting in lymphadenopathy. These findings support the importance of apoptotic regulation of lymphocyte persistence after antigen encounter in vivo.     

Murine lupus in MRL/lpr mice lacking CD4 or CD8 T cells

MRL/lpr mice develop a systemic autoimmune disease similar to systemic lupus erythematosus in humans. The mice show progressive lymphadenopathy due to the accumulation of an unusual population of CD4^?8^?(DN) B220⁺ αβ⁺ T cells. We bred MRL/lpr mice with mice lacking CD4⁺ or CD8⁺ T cells by gene targeting via homologous recombination in embryonal stem cells to determine the roles of these cells in the autoimmune disease. No difference in survival or autoantibody levels was noted between CD8-/- lpr and littermate controls. Interestingly, these CD8-/- lpr mice have a reduced level of B220⁺ DN T cells despite the fact that the degree of lymphadenopathy was unaltered. CD4-/- lpr mice had a diminished autoimmune disease with a reduction in autoantibody production and skin vasculitits, and increased survival compared to littermate controls. However, CD4-/- lpr mice had an enhanced splenomegaly that developed massively by 16–20 weeks of age (5 to 8 greater than lpr control mice) due to the accumulation of DN B220⁺ T cells. In addition, there were no differences in peripheral lymph node enlargement, although the proportion of DN B220⁺ T cells was about twofold higher in the CD4-/- lpr mice. These cells were phenotypically identical to the DN population in control lpr mice, indicating that the accumulating DN T cells can be dissociated from the autoimmune disease in these mice. Collectively, our results reveal that the autoimmune disease is dependent on CD4⁺, but not CD8⁺ T cells, and that many of the B220⁺ DN T cells traverse a CD8 developmental pathway.     

Characterization of NK cells and extrathymic T cells generated in the liver of irradiated mice with a liver shield

We previously reported that c-kit⁺ stem cells which give rise to extrathymic T cells are present in the liver of adult mice. Further characterization of extrathymic T cells in the liver of adult mice is conducted here. When mice with a liver shield were lethally (9.5 Gy) irradiated, all mice survived. All tested organs showed a distribution pattern of hepatic lymphocytes on day 7. The distribution pattern in the liver was characterized by an abundance of NK (CD3^? IL-2Rβ⁺) and extrathymic T cells (CD3^int IL-2Rβ⁺) before and after irradiation. To determine their function, post-irradiation allogeneic bone marrow transplantation (BMT) was performed in mice with or without a liver shield. Allogeneic BM cells were rejected in mice with a liver shield and specific activation of CD8⁺ CD3^int IL-2Rβ⁺ cells was induced. At that time, potent cytotoxicity of liver mononuclear cells (MNC) against allogeneic thymocytes was induced. Both NK1.1⁺ and NK1.1^? subsets of CD3^int cells expanded in these mice. An in vivo elimination experiment of the subsets indicated that the NK1.1⁺ subset of CD3^int cells (i.e. NK T cells) was much more associated with the rejection of allogeneic BM cells. However, even after the elimination of NK T cells, allogeneic BM cells were rejected. In this case, granulocytes expanded in parallel with NK1.1^? subsets. Granulocytes may also be associated with the rejection of allogeneic BM cells. These results suggest that the liver is an important haematopoietic organ even in adult life.     

CD8/CD4 lineage commitment occurs by an instructional/default process followed by positive selection

In the present study, we investigated the developmental potential of purified populations of transitional CD4ⁱⁿ CD8^hi and CD4^hi CD8ⁱⁿ thymocytes that were further defined according to their differentiation stage by their levels of T cell receptor (TCR) expression into TCR^lo, TCRⁱⁿ and TCR^hi subpopulations. The differentiation potential of each of these subsets was tested in vitro in a single-cell suspension culture assay that showed that CD4ⁱⁿ CD8^hi TCR^hi are precursors of CD8 single-positive cells, whereas CD4^hi CD8ⁱⁿ TCR^in/hi are precursors of both CD4 and CD8 single-positive thymocytes. The analysis of transitional subsets in mutant mice for either β2-microglobulin or major histocompatibility complex (MHC) class II further revealed that lineage commitment to the CD8 lineage requires a TCR-MHC class I engagement, presumably at the immature double-positive stage of thymic development, while CD4 commitment does not require an MHC class II-mediated signal, but rather occurs by default. Using the addition of MHC class I- or class II-expressing cells or the addition of total thymocytes to purified sorted transitional precursors for the duration of the cultures in vitro, we identified an additional stage of differentiation for both CD4 and CD8 lineages that requires a positive selection signal. Examination of protein tyrosine phosphorylation of transitional precursors revealed that CD4ⁱⁿ CD8^hi transitional cells contain a high level of a 70-kDa phosphorylated protein consistent with a role for ZAP70 in the signal transduction during the positive selection of CD8⁺ cells.     

The role of the interleukin-2 (IL-2)/IL-2 receptor pathway in MRL/lpr lymphadenopathy: The expanded CD4−8− T cell subset completely lacks functional IL-2 receptors

Autoimmune MRL/MP-lpr/lpr (MRL/lpr) mice spontaneously develop a systemic lupus erythematosus-like disease accompanied by a profound lymphadenopathy that consists of CD4^?8^?B220⁺ a P T cells. By the use of cross-linking experiments with radiolabeled interleukin-2 (IL-2), these abnormal T cells have been reported to constitutively express the IL-2 receptor β chain (IL-2Rα), a signal transducing component of IL-2R, in the absence of the a chain (IL-2Rα).To critically reevaluate the role of the IL-2/IL-2R pathway in the pathogenesis of lymphadenophathy we examined expression of the IL-2Rα and IL-2Rβ in MRL/lpr mice by ¹²⁵I-IL-2 binding analysis and also by flow cytometric analysis using monoclonal antibodies against each component of the receptor. We found that, contrary to the previous report, the CD4^?8^?B220⁺ α β T cells in lymph node (LN) of MRL/lpr mice were negative for both IL-2Rα and IL-2Rβ expression. The lpr liver CD4^?8^?B220⁺ a P T cells that had been implicated in the genesis of these abnormal LN T cells were also negative for IL-2Rβ expression. Therefore, our results indicate that the IL-2/IL-2R system plays little role, if any, in the expansion of abnormal CD4^?8^? B220⁺ α β T cells in MRL/lpr mice.