TCRint Cells 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.

PROTECTION AGAINST MALARIA DUE TO INNATE IMMUNITY ENHANCED BY LOW-PROTEIN DIET

Mice were fed ad libitum with a normal diet (25% protein) or low-protein diets (0-12.5% protein) for a wk and then infected with a nonlethal or lethal strain of Plasmodium yoelii, that is, blood stage infection. The same diet was continued until recovery. Mice fed with a normal diet showed severe parasitemia during nonlethal infection, but survived the infection. They died within 2 wk in the case of lethal infection. However, all mice fed with low-protein diets survived without apparent parasitemia (there were small peaks of parasitemia) in cases of both nonlethal and lethal strains. These surviving mice were found to have acquired potent innate immunity, showing the expansion of NK1.1 -TCRint cells and the production of autoantibodies during malarial infection. Severe combined immunodeficiency (scid) mice, which lack TCRint cells as well as TCRhigh cells, did not survive after malarial infection of lethal strain of P. yoelii, even when low-protein diets were given. These results suggest that low-protein diets enhanced innate immunity and inversely decreased conventional immunity, and that these immunological deviations rendered mice resistant against malaria. The present outcome also reminds us of our experience in the field study of malaria, in which some inhabitants eventually avoided contracting malaria even after apparent malarial infection.

Generation of B220low B cells and production of autoantibodies in mice with experimental amyloidosis: association of primordial T cells with this phenomenon.

To investigate the immunological state in amyloidosis, mice were twice intraperitoneally injected (2-week interval) with casein emulsified in complete Freund's adjuvant. Two weeks after the treatment, amyloid deposits were detected in the spleen and other organs of these mice. The number of lymphocytes yielded by the liver and spleen increased significantly. The most affected lymphocyte subset was found to be B cells, namely, the total number of B cells increased and unusual B220low B cells were newly generated in the liver and spleen. In other words, not only normal B220high B cells but also unusual B220low B cells were detected in these organs of mice with amyloidosis. In parallel with this phenomenon, autoantibodies against denatured DNA were detected in sera. Since such autoantibodies are known to accompany the functional activation of NKT cells, NKT cell-deficient mice were used for the induction of amyloidosis. Such mice showed less formation of amyloidosis and lower levels of autoantibodies in sera. Athymic nude mice were NKT cell-deficient but NK1.1- TCRint cells were present. These athymic mice showed an intermediate induction of amyloidosis. The cytokine profile seen in mice with amyloidosis was the Th0 type, showing simultaneous production of IL-4 and IFNgamma. These results suggest that the generation of B220low B cells and the production of autoantibodies in aid of primordial T cells may be major immunological mechanisms in amyloidosis mice.

Estimation of effector cytotoxic lymphocytes against male H-Y antigens induced by two-step stimulations as CD8+NK1.1-TCRint and CD8+NK1.1+TCRint cells

C57BL/6 (B6) female mice were injected with 107 splenocytes of B6 male mice to immunize male H-Y antigens. This stimulation induced a slight increase in the proportions of CD3intIL-2Rβ+ cells, CD3intNK1.1+ cells (i.e., NKT cells), and CD4+T cells in the liver and spleen. However, cytotoxic activity against male H-Y antigens was not detected in hepatic and splenic lymphocytes. When these in vivo primed hepatic or splenic lymphocytes were cultured in vitro with irradiated splenocytes of male mice (i.e., mixed lymphocyte reaction, MLR), potent cytotoxicity against male H-Y antigens was induced. Unprimed (in vivo) hepatic and splenic lymphocytes did not acquire such activity even by in vitro stimulation. If the primed mice were in vivo stimulated again with male lymphocytes, such cytotoxicity was not obtained. In other words, in vivo priming and in vitro culture stimulation were both required for the induction of cytotoxicity. Phenotypic characterization and cell-depletion study using normal B6 mice and NKT-deficient mice revealed that effector cells were both CD8+NK1.1−TCRint and CD8+NK1.1+TCRint cells. These results suggest that recognition of H-Y antigens and effector function against H-Y antigens may be events of innate immunity similar to the case of other self-related antigens.

Essential role of extrathymic T cells in protection against malaria.

Athymic nude mice carry neither conventional T cells nor NKT cells of thymic origin. However, NK1.1(-)TCR(int) cells are present in the liver and other immune organs of athymic mice, because these lymphocyte subsets are truly of extrathymic origin. In this study, we examined whether extrathymic T cells had the capability to protect mice from malarial infection. Although B6-nu/nu mice were more sensitive to malaria than control B6 mice, these athymic mice were able to survive malaria when a reduced number of parasitized erythrocytes (5 x 10(3) per mouse) were injected. At the fulminant stage, lymphocytosis occurred in the liver and the major expanding lymphocytes were NK1.1(-)TCR(int) cells (IL-2Rbeta(+)TCRalphabeta(+)). Unconventional CD8(+) NKT cells (V(alpha)14(-)) also appeared. Similar to the case of B6 mice, autoantibodies (IgM type) against denatured DNA appeared during malarial infection. Immune lymphocytes isolated from the liver of athymic mice which had recovered from malaria were capable of protecting irradiated euthymic and athymic mice from malaria when cell transfer experiments were conducted. In conjunction with the previous results in euthymic mice, the present results in athymic mice suggest that the major lymphocyte subsets associated with protection against malaria might be extrathymic T cells.

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.

U1B3.3 monoclonal antibody recognizes a precursor of NK1.1<sup>+</sup> cytotoxic T cells generated by interleukin-2

U1B3.3 monoclonal antibody (mAb) was established by immunizing a rat with the tMK-2U lymphoma cell line, derived from athymic nude mice. U1B3.3 mAb recognizes some T cell populations, B cells and natural killer (NK) cells. U1B3.3+ T cells express CD3/T cell receptor (TCR) complex at a unique intensity: between that of TCR-intermediate cells (TCRint cells) and TCRbright cells. TCRint cells strongly express the interleukin-2 receptor β chain (IL-2Rβ), but almost all U1B3.3+ T cells express high or low levels of IL-2Rβ. When purified U1B3.3+ NK1.1-T cells were cultured with IL-2, approximately 14% of the cells acquired expression of NK1.1 molecules, which are expressed on NK cells and natural killer T (NKT) cells. Furthermore, U1B3.3+ NK1.1-T cells killed tumor cells after culture with IL-2. These results indicate that U1B3.3 mAb recognizes a precursor of NK1.1+ cytotoxic T cells generated by IL-2.

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.

TCRhigh cells are generated by the mainstream of T cell differentiation in the thymus, whereas TCRint 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 TCRint cells with TCRhigh cells, we used congenitally athymic nude (B6-nu/nu) mice which carry only TCRint cells in all immune organs. When TCRhigh cells from B6-C-H-2bm12 (bm12) mice (i.e. I-Abm12) were injected into B6-nu/nu mice (i.e. 1-Ab), the expanding T cell population was a mixture of TCRhigh cells of donor origin and TCRint cells of recipient origin. However, 9 Gy-irradiated nude mice permitted a full expansion of TCRhigh cells which expressed the IL-2Ralpha+beta+ 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 TCRhigh cells of donor origin and such TCRhigh cells continued to have the IL-2Ralpha(+/-)beta+ phenotype. These mice could survive but showed signs of chronic GVHD thereafter. In both situations, CD4+alphabeta T cells expanded irrespective of donor or recipient origin. These results suggest that TCRint cells in the recipient mice possess a regulatory function in relation to donor TCRhigh cells; as a result, fully activated TCRhigh cells acquired the IL-2Ralpha+beta+ phenotype and injured the host, but TCRhigh cells suppressed in vivo remained as the IL-2Ralpha(+/-)beta+ phenotype and only partially injured the host.

Mice with early onset of death (EOD) due to lupus glomerulonephritis.

Both MRL-lpr/lpr (lpr) and BXSB mice fall victim to autoimmune disease as a function of age. To combine their properties, brother-sister mating of (female lpr x male BXSB)F1 mice was done. Mice for mating were selected according to indicators of early onset of glomerulonephritis and subsequent early death (i.e., EOD). This mating was continued for more than 16 generations. The EOD mice thus established had homozygous H-2k/k, lpr/lpr, and possible yaa/- (in the case of males). The average life span of males was 83 days while that of females was 126 days. After 12 weeks of age, the majority (> 80%) of male EOD mice were characterized by the abnormality of urine due to glomerulonephritis. We then characterized how glomerulonephritis was evoked, especially in terms of expanding lymphocyte subsets in various immune organs. Similar to the case of parental lpr mice, the major expanding cells were CD4-8-B220+ TCRint cells in the immune organs and kidney. In addition, myeloid cells were found to infiltrate the kidney. This massive infiltration of both TCRint cells and myeloid cells might be responsible for the onset of acute glomerulonephritis. Even after more than 50 generations, these EOD mice still carry both lpr and yaa genes. These results suggest that EOD mice might be a very useful tool for the study of acute lupus glomerulonephritis which is evoked by the genetic abnormalities.

Quick recovery in the generation of self-reactive CD4low natural killer (NK) T cells by an alternative intrathymic pathway when restored from acute thymic atrophy.

The thymus comprises the mainstream of T cell differentiation which produces conventional T cells and an alternative pathway which produces primordial T cells with intermediate density of T cell receptor (TCR)-CD3 complex on the surface (i.e. intermediate TCR cells or TCRint cells). We induced acute thymic atrophy in mice by an administration of hydrocortisone (10 mg) or irradiation (6.5 Gy). It was demonstrated that CD3intCD4lowNK1.1+ T cells were immediately generated by an alternative intrathymic pathway without passing through the double-positive CD4+8+ stage, when restored from thymic atrophy (days 3-14). These CD3intCD4lowNK1.1+ T cells mediated self-reactivity and appeared even in the periphery. mRNA of an invariant chain of TCR Valpha14Jalpha281 gene product was detected in these CD4low T cells, but not remaining CD4high T cells. The mainstream of T cell differentiation in the thymus was not restored up to day 14 and there was no leakage of self-reactive clones into the population generated through the mainstream. These results reveal that an alternative intrathymic pathway is associated with the generation of self-reactive T cells, in an early restoration phase after thymic atrophy.

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.

Association of Intermediate T Cell Receptor Cells, Mainly Their NK1.1− Subset, with Protection from Malaria

Mice were infected with Plasmodium (P.) yoelii blood-stage parasites. Both the liver and spleen were the sites of inflammation during malarial infection at the beginning of day 7. The major expanding cells were found to be NK1.1− intermediate αβTCR (αβTCRint) in the liver and spleen, although the population of NK1.1+ αβTCRint cells remained constant or slightly increased. These TCRint cells are of extrathymic origin or are generated by an alternative intrathymic pathway and are distinguished from conventional T cells of thymic origin. During malarial infection, the population of conventional T cells did not increase at all. TCRint cells purified from the liver of mice which had recovered from P. yoelii infection protected mice from malaria when they were transferred into 6.5-Gy-irradiated mice. Interestingly, the immunity against malaria seemed to disappear as a function of time after recovery, namely, mice which had recovered from malaria 1 year previously again became susceptible to malarial infection. The present results suggest that TCRint cells are intimately associated with protection against malarial infection and, therefore, that mice which had recovered from malaria 1 year previously lost such immunity.

Consistent infiltration of thymus-derived T cells into the parenchymal space of the liver in normal mice.

We previously reported that extrathymic T cells (intermediate T-cell receptor cells [TCR(int) cells]) are in situ generated in the parenchymal space of the liver in mice. They subsequently migrate to the sinusoidal lumen. In this study, we characterized how such extrathymic T cells, natural killer (NK) cells, and thymus-derived T cells (high T-cell receptor cells [TCR(high) cells]) localized in the parenchymal space or the sinusoidal lumen of mice. To this end, liver irrigation with physiological saline from the portal vein was performed and the distribution of lymphocyte subsets was compared between the liver (i.e., lymphocytes in the parenchymal space) and the irrigation solution (i.e., lymphocytes in the sinusoidal lumen). Extrathymic T cells and NK cells were found to be abundant in both the liver and sinusoidal lumen. As expected, thymus-derived T cells were abundant in the sinusoidal lumen. However, a significant proportion of thymus-derived T cells were always present in the parenchymal space, even after intensive irrigation with or without collagenase. These results suggest that thymus-derived T cells may consistently infiltrate the parenchymal space from the sinusoidal lumen in normal mice. This possibility was confirmed by (1) the injection of B6 splenic cells (TCR(high) cells) or the thymus graft into B6-nu/nu mice (presence of only TCR(int) cells) and by (2) using parabiotic mice of B6.Ly5.1 and B6.Ly5.2 strains (sharing circulation) in conjunction with immunofluorescence tests and immunohistochemical staining. In other words, inverted routes of migration and homing between extrathymic T cells and thymus-derived T cells exist in the liver.

Characterization of subpopulations of T-cell receptor intermediate (TCRint) T cells.

CD1-autoreactive T cells of two types have been demonstrated among T cells expressing the T-cell receptor (TCR) alphabeta at intermediate levels (TCRint cells). One type constitutes a major fraction of the natural killer (NK)1.1+ TCRint population in C57BL/6 (B6) mice and carries a restricted TCR composed of an alpha-chain with an invariant Valpha14-J281 rearrangement, and a beta-chain using Vbeta8. 2, 7 or 2. The second type utilises a variety of TCR and was derived from CD4+ cells in mice lacking MHC class II. To increase our understanding of the two different CD1-reactive subsets, we have investigated and compared the populations of origin: NK1.1+ and NK1. 1- TCRint subsets from MHC class II-deficient mice and CD4+NK1.1+ T cells from B6 mice. The three TCRint populations shared a phenotype indicating previous activation, and contained low frequencies of cells expressing NK receptors of the Ly49 family. In contrast to control CD4+ cells, the three TCRint subsets produced high amounts of interleukin (IL)-4 and interferon (IFN)-gamma after activation. Importantly, no IL-10 could be detected in either TCRint population, implying a distinct function for these cells, different from those of conventional CD8+ and CD4+ cells, including the typical T-helper 2 (Th2) cell. Analysis of TCR expression indicated that the proportion of cells using the semi-invariant Valpha14/Vbeta8.2-type TCR was lower in NK1.1+ cells from MHC class II-negative mice than in CD4+NK1.1+ B6 cells. Further, usage of the Valpha14-J281 rearrangement was also demonstrated among NK1.1- TCRint cells.

Splenic NK1.1-Negative, TCRαβ Intermediate CD4+ T Cells Exist in Naive NK1.1 Allelic Positive and Negative Mice, with the Capacity to Rapidly Secrete Large Amounts of IL-4 and IFN-γ Upon Primary TCR Stimulation

AbstractSplenic NK1.1+CD4+ T cells that express intermediate levels of TCRαβ molecules (TCRint) and the DX5 Ag (believed to identify an equivalent population in NK1.1 allelic negative mice) possess the ability to rapidly produce high quantities of immunomodulatory cytokines, notably IL-4 and IFN-γ, upon primary TCR activation in vivo. Indeed, only T cells expressing the NK1.1 Ag appear to be capable of this function. In this study, we demonstrate that splenic NK1.1-negative TCRintCD4+ T cells, identified on the basis of FcγR expression, exist in naive NK1.1 allelic positive (C57BL/6) and negative (C3H/HeN) mice with the capacity to produce large amounts of IL-4 and IFN-γ after only 8 h of primary CD3 stimulation in vitro. Furthermore, a comparison of the amounts of early cytokines produced by FcγR+CD4+TCRint T cells with NK1.1+CD4+ or DX5+CD4+TCRint T cells, simultaneously isolated from C57BL/6 or C3H/HeN mice, revealed strain and population differences. Thus, FcγR defines another subpopulation of splenic CD4+TCRint cells that can rapidly produce large concentrations of immunomodulatory cytokines, suggesting that CD4+TCRint T cells themselves may represent a unique family of immunoregulatory CD4+ T cells whose members include FcγR+CD4+ and NK1.1/DX5+CD4+ T cells.

Oligoclonality of TCRint cells with a low diversity of TCR complementarity-determining region 3 in mice with graft-versus-host disease.

Conventional T cells (i.e. TCRhigh) are generated by the main stream of T-cell differentiation in the thymus. However, primordial T cells (i.e. TCRint) are generated by extrathymic pathways and an alternative intrathymic pathway. Since TCRint cells contain self-reactive clones, the diversity of the T-cell antigen receptor (TCR) complementarity-determining region (CDR) 3 was examined. The predominant Vbeta8.2+ clones among TCRint cells were selected for DNA sequencing. Thymectomized, irradiated mice subjected to bone-marrow transplantation (BMT) were used; graft-versus-host disease (GVHD), B6-->(B6xC3H/He)F1 and syngeneic BMT, B6-->B6. In these combinations, only TCRint cells were generated. Vbeta8.2+ cells with a low diversity of CDR3 of V-gene expanded in GVHD mice. Vbeta8.2+ cells of TCRint and TCRhigh cells in normal mice were polyclonal, showing that the former has a lower diversity of CDR3 than the latter. The clonality of activated TCRhigh cells was examined, in which CD3high cells (bml2 mice) were injected into 1 Gy-irradiated B6 nude mice. Some Vbeta8.2+ clones among TCRhigh cells were expanding but the diversity of CDR3 was greater than that of CD3int cells, despite the fact that the recognition site of the H-2 difference was smaller. Taken together with invariant usage of V alpha14, these results suggest that TCRint cells have a low diversity of CDR3 of Vbeta genes.

An allogeneic microenvironment influences the phenotype of intermediate T-cell receptor cells expanding in MRL-lpr/lpr mice.

MRL-lpr/lpr (lpr) mice fall victim to autoimmune disease owing to a lymphoproliferative disorder mainly of double-negative (DN) CD4- CD8- alpha beta T cells expressing a low density of interleukin-2 receptor beta-chain (IL-2R beta). It was previously revealed that the lpr gene is a defective Fas gene, into which an early transposon (ETn) of retrovirus is transfected. As a result of the failure of apoptosis, intermediate T-cell receptor (TCR) cells (i.e. TCRint cells) with DN phenotype abnormally accumulate in the periphery of lpr mice. We investigated herein how these TCRint cells are selected in terms of CD4, CD8 and TCR in lpr mice. When a whole fraction of mononuclear cells (MNC) in various immune organs of lpr mice was injected into scid mice (allogeneic circumstance), CD8+ TCRint cells mainly expanded. They had a high density of IL-2R beta. This was true when bone marrow cells of lpr mice were injected into scid mice. On the other hand, when MNC of the spleen and bone marrow in lpr mice were injected into irradiated (9 Gy) lpr mice (syngeneic circumstance), the major expanding cells were DN TCRint cells expressing a low density of IL-2R beta. A cell-sorting experiment for purified fractions demonstrated that only CD8- cells reconstituted TCRint cells in scid mice. Namely, DN CD4- CD8- cells as well as CD4+ cells which once acquired the mature phenotype, no longer switched their phenotype. These results suggest that the phenotype of TCRint cells is influenced by the surrounding microenvironment.

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.

Autologous killing by a population of intermediate T-cell receptor cells and its NK1.1+ and NK1.1- subsets, using Fas ligand/Fas molecules.

Self-reactive clones, estimated by anti-V beta monoclonal antibodies (mAb) in conjunction with the Mls system, are confined to a population of intermediate (int) T-cell receptor (TCR) (or CD3) cells (i.e. TCRint cells), but are not found among TCRhigh cells. The next questions to be answered are whether autologous killing is confined to TCRint cells and how such killing is mediated. In this study, 51Cr-labelled thymocytes of syngeneic or allogeneic origin were used as target cells (4-hr assay). When liver and splenic mononuclear cells (MNC) obtained from B6 mice were used as effector cells, prominent autologous killing was seen in liver MNC, but not splenic MNC. Such killing was not seen when thymocytes from B6-lpr/lpr mice (i.e. Fas-) were used as target cells, nor when liver MNC from MRL-gld/gld mice (i.e. Fas ligand-) were used as effector cells (target thymocytes of MRL(-)+/+ mice). Cell separation experiments using a cell sorter revealed that autologous killing was mediated for the most part by CD3int cells, while allogeneic killing was mediated entirely by natural killer (NK) cells, TCRint cells and TCRhigh cells. Among CD3int cells, the NK1.1+ subset (i.e. NK1.1+ T cells) manifested a higher level of autologous killing than did the NK1.1- subset. Consistent with the results of a functional assay, it was found by reverse-transcription-polymerase chain reaction (RT-PCR) assay that CD3int cells among liver MNC showed the expression of Fas ligand mRNA, while thymocytes expressed Fas mRNA. When class I major histocompatibility complex (MHC)- thymocytes (from beta 2-microglobulin-deficient mice) were used as target cells, NK cells, but not CD3int cells, showed potent cytotoxicity. These results suggest that autologous killing is a major function of TCRint cells with self-reactivity, and that such killing is mediated by means of Fas ligand/Fas molecules.