TCR Int Research Articles

Identification and characterization of autoantibody-producing B220 low B (B-1) cells appearing in malarial infection

Mice with malaria showed unique immunological responses, including the expansion of NK1.1 −TCR int cells (extrathymic T cells). Since TCR int cells with autoreactivity and autoantibody-producing B cells (B-1 cells) are often simultaneously activated under autoimmune conditions, it was examined whether B-1 cells were activated in the course of malarial infection. From days 14 after infection, B220 low B-1 cells appeared in the liver and spleen. The number of B220 low B cells was highest at day 14, but the ratio was highest at days 28–35. In parallel with the appearance of B220 low cells, autoantibodies against HEp-2 cells and double-stranded DNA were detected in sera. These B220 low cells had phenotypes of CD44 high, CD23 − and CD62L −. In sharp contrast, conventional B220 high B cells (B-2 cells) were CD44 low, CD23 + and CD62L +. These results suggested that malaria immune responses were not mediated by conventional T and B cells but resembled the responses during autoimmune diseases.

Identification of effector cells for TNFα-mediated cytotoxicity against WEHI164S cells

WEHI164S cells were found to be very sensitive targets for in vitro killing in a 6-h culture when liver or splenic lymphocytes were used as effector cells in mice. Of particular interest, a limiting cell-dilution analysis showed that effector cells were present in the liver with a high frequency (1/4300). In contrast to YAC-1 cells as NK targets, perforin-based cytotoxicity was not highly associated with WEHI164S killing. The major killer mechanism for WEHI164S targets was TNFα-mediated cytotoxicity. By cell sorting experiments, both NK cells and intermediate T cells (i.e., TCR int cells) were found to contain effector cells against WEHI164S cells. However, the killer mechanisms underlying these effector cells were different. Namely, NK cells killed WEHI164S cells by perforin-based cytotoxicity, TNFα-mediated cytotoxicity, Fas ligand cytotoxicity, and other mechanisms, whereas intermediate T cells did so mainly by TNFα-mediated cytotoxicity. These results suggest that TNFα-mediated cytotoxicity mediated by so-called natural cytotoxic (NC) cells comprised events which were performed by both NK and intermediate T cells using somewhat different killer mechanisms. Intermediate T cells which were present in the liver were able to produce TNFα if there was appropriate stimulation.

Resistance to Malarial Infection Is Achieved by the Cooperation of NK1.1+ and NK1.1− Subsets of Intermediate TCR Cells Which Are Constituents of Innate Immunity

We previously reported that the major expanding lymphocytes were intermediate TCR (TCRint) cells (mainly NK1.1−) during malarial infection in mice. Cell transfer experiments of TCRint cells indicated that these T cells mediated resistance to malaria. However, TCRint cells always contain NK1.1+TCRint cells (i.e., NKT cells) and controversial results (NKT cells were effective or not for resistance to malaria) have been reported by different investigators. In this study, we used CD1d(−/−) mice, which almost completely lack NKT cells in the liver and other immune organs. Parasitemia was prolonged in the blood of CD1d(−/−) mice and the expansion of lymphocytes in the liver of these mice was more prominent after an injection of Plasmodium yoelii-infected erythrocytes. However, these mice finally recovered from malaria. In contrast to B6 mice, CD4−8− NKT cells as well as NK1.1−CD3int cells expanded in CD1d(−/−) mice after malarial infection, instead of CD4+ (and CD8+) NKT cells. These newly generated CD4−8−NKT cells in CD1d(−/−) mice did not use an invariant chain of Vα14Jα281 for TCRα. Other evidence was that severe thymic atrophy and autoantibody production were accompanied by malarial infection, irrespective of the mice used. These results suggest that both NK1.1− and NK1.1+ subsets of TCRint cells (i.e., constituents of innate immunity) are associated with resistance to malaria and that an autoimmune-like state is induced during malarial infection.

Intensive generation of NK1.1- extrathymic T cells in the liver by injection of bone marrow cells isolated from mice with a mutation of polymorphic major histocompatibility complex antigens.

Whether intermediate TCR (TCRint) cells and natural killer T (NKT or NK1.1+TCRint) cells are extrathymically generated remains controversial. This arises from the fact that there are few of these T cells in athymic nude mice and neonatally thymectomized mice. However, when athymic mice were provided with appropriate microenvironments or stimulation, many TCRint cells (mainly NK1.1-) were found to arise in the liver. NKT cells are known to be positively selected by monomorphic major histocompatibility complex (MHC) -like antigens (e.g. CD1d). This is true even if they are CD4+. In other words, a MHC class I-like antigen is restricted to CD4 antigen. This rule is somewhat different from that seen in conventional T cells (i.e. the restriction of class II with CD4 and that of class I and CD8). In the case of NK1.1-TCRint cells, they were selected by polymorphic MHC antigens, but their MHC restriction to CD4 or CD8 antigen was incomplete. This was revealed by experiments of bone marrow transfer with class I (bm 1) or II (bm 12) disparity. Depending on the disparity, a unique cytokine profile in sera was detected. These results suggest that the development of T lineage lymphocytes and MHC restriction to CD4 and CD8 might have occurred in parallell as a phylogenic event, and that NK1.1- extrathymic T cells (i.e. NK1.1-TCRint) are at an intermediate position between NKT cells and conventional T cells in phylogeny.

Mouse NK1.1 + cytotoxic T cells can be generated by IL-2 exposure from lymphocytes which express an intermediate level of T cell receptor

NK-like T cells which express the NK1.1 molecule and CD3 (or TCR) of intermediate level (CD3 int or TCR int cells) were recently demonstrated to be present in various immune organs, and to have NK-like cytotoxic activity against NK target cells. In this study, we investigated whether NK1.1 − T cells could express NK1.1. We found that NK1.1 + TCR int cells were much more abundant in the liver (20%) than in the spleen (2%). When hepatic and splenic mononuclear cells (MNCs) were cultured either in the absence of IL-2 or in the presence of CD3/TCR cross-linking, the original NK1.1 + TCR int cells disappeared. However, when they were cultured in the presence of a high dose of IL-2 for 4 days, a new type of NK1.1 + T cell was formed to the extent of approximately 15–20%, and the liver and spleen contained similar percentages of this new type of NK1.1 + T cells. The phenotypes of the original and the new type of NK1.1 + T cells were clearly distinct. The freshly obtained NK1.1 + TCR int cells consisted of double-negative (DN) CD4 −CD8 − cells and single-positive (SP) CD4 + cells, whereas the new type of NK1.1 + T cells predominantly consisted of DN CD4 −CD8 − cells and SP CD8 + cells and expressed a high level of CD3 (CD3 high or TCR high cells). When NK1.1 − cells or IL-2 receptor β-chain (IL-2R β) − cells were isolated from the liver and spleen, and cultured in the presence of IL-2 for 4 days, NK1.1 + T cells were generated from NK1.1 − cells, but not from IL-2R β − cells. Our results suggested that the NK1.1 − cells, but not IL-2R β − cells, contained the precursor of IL-2-stimulated NK1.1 + TCR high cells. When purified NK1.1 − IL-2R β + TCR int cells were cultured in the presence of IL-2 for 4 days, approximately 10% of the cells became NK1.1 + TCR high cells. Approximately 60% of the purified NK1.1 + TCR int cells lost NK1.1 expression. The IL-2-stimulated NK1.1 + TCR high cells that had arisen from NK1.1 − TCR int cells exerted an NK cell-like cytotoxic activity similar to that of the original NK1.1 + T cells. Thus, NK1.1 − TCR int cells could express NK1.1 and exert NK-like cytotoxic activity regardless of their origin. It appears that NK1.1 + TCR high cells can only be induced through an IL-2-stimulation pathway but not via CD3/TCR cross-linking.

Absolute dependence of T cell receptor(hi) cell generation and relative dependence of T cell receptor(int) 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. TCRhi). 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. TCRint). 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 TCRhi cells de novo in the periphery as well as in the grafted thymi, and the absolute number of interleukin-2 receptor beta chain+ TCRint cells increased prominently in number in the periphery. Among thymic hormones tested, the administration of thymosin alpha induced a slight expansion of CD3int cells in nude mice. To examine a possible interaction of TCRint cells with TCRhi cells in the periphery, B6 nu/nu mice (Ly5.2+) were injected with TCRhi cells purified from the spleen of B6 Ly5.1 congenic mice. In this case, TCRint (Ly5.2+) cells expanded well in all tested organs of nude mice. These results suggest that the generation of TCRhi cells is absolutely dependent on the thymus and that TCRint cells expand under the influence of the thymus (humoral) and due to interaction with thymus-derived conventional T cells.

LPS induces NK1.1+ alpha beta T cells with potent cytotoxicity in the liver of mice via production of IL-12 from Kupffer cells.

We recently reported that systemic administration of IL-12 into mice activates NK1.1+ alpha beta T cells with intermediate TCR (NK1+TCRint) and induces strong MHC-unrestricted cytotoxicity in C57BL/6 mice. In the present report, we examined the effect of LPS on Kupffer cells and NK1+TCRint, cells in C57BL/6 mice. Administration of LPS, as well as synthetic lipid A analogue (ONO-4007), but not detoxified LPS, induces the increase of NK1 expression of NK1+TCRint cells (NKlhighTCRint) and the acquisition of strong MHC-unrestricted cytotoxicity of these cells against NK-sensitive and NK-resistant targets as does IL-12 administration. LPS as well as ONO-4007 induced IL-12 mRNA in hepatic mononuclear cells, mainly in plastic-adherent Kupffer cells. LPS-induced cytotoxicity of hepatic mononuclear cells was greatly reduced by in vivo injections of anti-IL-12 Ab, to a lesser extent by anti-IFN-gamma Ab, but not by anti-IL-1 nor anti-TNF-alpha Ab. Pretreatment of mice with LPS induced inhibition of hepatic metastases of i.v. injected EL4 cells in C57BL/6 euthymic and athymic mice and this antimetastasis was inhibited by injection of anti-IL-12 Ab. This antimetastatic effect of LPS in the liver was also observed in different strains of mice and tumors, In contrast to IL-12, however, LPS was not so effective when administered after tumor inoculation. These results revealed that LPS (lipid A) stimulates NK1+TCRint cells through IL-12 production from Kupffer cells and suggest that bacterial components, probably including those from intestine, are activators of Kupffer cells and NK1+TCRint, cells in the liver. It is also suggested that the host condition as well as LPS-induced cytokines other than IL-12 may affect antitumor effect induced by LPS in the liver.

CD4low TCRint thymocytes do not belong to the CD8 lineage maturation pathway.

Thymocytes with a low expression of CD4 and an intermediate density of the TCR (CD4low TCRint) were analyzed for phenotype, MHC dependence, production kinetics, and TCR repertoire to investigate their position in the intrathymic T cell maturation process. Comparison of normal and MHC-deficient mice showed that the CD4low TCRint cell subset was MHC class II dependent, as this subpopulation could not be defined in MHC class II- or double (class I and II)-deficient mice. These thymocytes were heat-stable Aghigh and CD69+, thus immature and recently engaged in a TCR interaction, probably with MHC class II molecules. Their generation kinetics were studied in two systems: development of exogenous bone marrow cells transferred into RAG-2-/- mice, and pulse labeling with bromodeoxyuridine. In both systems, CD4low TCRint cells were produced well before CD4low TCRhigh cells, the direct precursors of CD8 single-positive cells. Their production paralleled that of CD4high TCRint cells, but they were different than these thymocytes in their smaller cell size. Moreover, they had the same V beta 6 frequency in Mls-1a and Mls-1b mice, suggesting that these cells could be undergoing a negative selection process. The data here clearly demonstrate that CD4low TCRint thymocytes do not belong to the CD8 lineage maturation pathway, and suggest that these cells could represent a MHC class II-restricted dead-end subset.

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 beta-chain (IL-2R beta) intermediate TCR (TCRint) cells by two-color staining for CD3 or T cell receptor (TCR) and IL-2R beta. The elimination of Kupffer cells did not significantly affect levels of TCRint 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, CD3int cells, but not other type of cells, decreased prominently. Purified CD3int 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 extra-thymic generation of TCRint cells and their acquisition of cytotoxic function are relatively independent of Kupffer cells, but are dependent on hepatocytes.

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 (TCRhigh) differentiate in the major intrathymic pathway and then distribute to the peripheral immune organs, whereas T cells expressing intermediate levels of the TCR (TCRint) differentiate in both extrathymic pathways and an alternative intrathymic pathway and localize in unique sites, including the liver and thymic medulla. Since TCRint cells constitutively express interleukin-2 receptor beta-chain (IL-2R beta), two-color staining for CD3 (or TCR) and IL-2R beta clearly distinguished IL-2R beta+ CD3int (or TCRint) cells from IL-2R beta-, CD3high cells. CD3int cells may be considered to be primordial T cells based on their phenotype, morphology and other functional properties. In this study, using anti-V beta 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-2R beta+, CD3int cells, in all of the organs tested. A significant proportion of self-reactive clones was never identified among CD3high 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, CD3int cells, but neither NK cells nor CD3high cells had a potent cytotoxic effect against a syngeneic hepatoma in the presence of anti-CD3 mAb. These results raise the possibility that CD3int 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.

Cytotoxic NK1.1 Ag+ alpha beta T cells with intermediate TCR induced in the liver of mice by IL-12.

Systemic administration of IL-12 greatly reduced the hepatic metastases of i.v.-injected liver metastatic EL4 tumor cells in C57BL/6 +/+ and nu/nu mice. Cytotoxic assay in vitro revealed that administration of IL-12 greatly enhanced cytotoxicity of hepatic mononuclear cells (MNC) against various NK- sensitive and -resistant tumor targets, including EL4 cells, whereas only slight or moderate augmentation of the cytotoxicity was observed in splenocytes in normal and nude mice. After IL-12 administration, hepatic MNC increased in number and showed vigorous proliferation in vitro. Hepatic MNC of control C57BL/6 +/+ mice contain alpha beta T cells with intermediate TCR (TCRint) as well as alpha beta T cells with bright TCR, whereas hepatic MNC of nu/nu mice have only TCRint cells. These TCRint cells are found to be NK1.1 Ag+ (NK1+ TCRint). Systemic administration of IL-12 into normal and nude mice markedly augments the NK1 expression of NK1+ TCRint cells (NK1high TCRint), which is comparable to or brighter than that of NK cells in the liver, whereas alpha beta T cells with bright TCR or gamma delta T cells in the liver are NK1-. Depletion of either NK1.1+ or CD3+ cells, but not CD8+ cells, of hepatic MNC from IL-12-treated normal mice by respective Abs and C in vitro abrogate their cytotoxicity. These results revealed that TCRint cells are potent cytotoxic effector cells and suggest that NK1high TCRint cells are the main antimetastatic population in the liver, and that TCRint cells are functionally different from regular T cells with bright TCR.