Negative regulation of expression and function of Fc gamma RIII by CD3 zeta in murine NK cells.

Fc gamma RIII is involved in Ab-dependent cell-mediated cytotoxicity (ADCC) and cytokine production by NK cells. Signaling and expression of Fc gamma RIII are dependent on FcR gamma. Although NK cells express not only FcR gamma but also CD3 zeta, the role of CD3 zeta in NK cell function remains unclear. Here, we found that the expression of Fc gamma RIII on NK cells from CD3 zeta-deficient mice is unexpectedly up-regulated compared with that on cells from normal mice. Furthermore, ADCC and IFN-gamma production upon Fc gamma RIII-cross-linking by NK cells from CD3 zeta-deficient mice were also up-regulated. Up-regulation of the surface expression of Fc gamma RIII on CD3 zeta-deficient NK cells is not mediated by transcriptional augmentation of either Fc gamma RIII or FcR gamma gene because there was no significant difference in the expression of mRNA for Fc gamma RIII and FcR gamma. Transfection of CD3 zeta into a cell line expressing Fc gamma RIII and FcR gamma induced a decrease in the cell surface expression of Fc gamma RIII. These findings reveal a negative regulatory role of CD3 zeta in Fc gamma RIII-mediated function of murine NK cells.

Resistance of Fc receptor- deficient mice to fatal glomerulonephritis.

Immune complex-mediated inflammation is a common mechanism of various autoimmune diseases. Glomerulonephritis (GN) is one of these diseases, and the main mechanism of the induction of GN has been unclear. We examined the contribution of Fc receptors in the induction of nephrotoxic GN by establishing and analyzing mice deficient in the Fc receptor gamma chain (FcRgamma). Whereas all wild-type mice died from severe glomerulonephritis with hypernitremia by administration of anti-glomerular basement membrane (GBM) antibodies, all FcRgamma-deficient mice survived. Histologically, wild-type mice showed glomerular hypercellularity and thrombotic changes, whereas the renal tissue in FcRgamma-deficient mice was almost intact. Deposition of anti-GBM antibody as well as complement components in the GBM were equally observed in both wild-type and knockout mice. These results demonstrate that the triggering of this type of glomerulonephritis is completely dependent on FcR+ cells.

Influence of graft versus host reaction on the T cell repertoire differentiating from bone marrow precursors following allogeneic bone marrow transplantation.

When lethally irradiated AKR (Mls-1a) mice were reconstituted with bone marrow (BM) cells plus a small number (0.5%) of mature T cells from allogeneic B10.AQR or B10 (Mls-1b) mice and minor GVHR was induced in the recipients, almost complete donor chimerism was accomplished in the early stages after reconstitution. By contrast, in irradiated AKR mice reconstituted with T cell-depleted BM cells alone from B10 or B10.AQR mice, radio-resistant T cells of recipient origin persisted for a relatively long period in peripheral lymphoid tissues. In this paper the influence of residual T cells in the chimeric mice on generation of the T cell repertoire derived from donor BM is discussed. It will be demonstrated that the recipient (AKR) T cells are capable of producing Mls-1a antigens (Ag) after lethal irradiation in vivo. These recipient T cells eventually induce clonal elimination of Mls-1a reactive V beta 6+, V beta 8.1+ and V beta 9+ T cells derived from developing thymocytes of donor BM origin. The Mls-1a reactive T cells are not eliminated in GVHR chimeras in which recipient T cells are absent. However, V beta 5+ T cells reactive to I-E plus Etc-1 Ag are deleted in the chimeras undergoing GVHR. These results indicate that recipient cells which produce tissue-specific antigens (tolerogens) should be taken into consideration when generation of the T cell repertoire of donor origin following allogeneic BM transplantation is investigated.

Association with FcRgamma is essential for activation signal through NKR-P1 (CD161) in natural killer (NK) cells and NK1.1+ T cells.

Natural killer (NK) cells exhibit cytotoxicity against variety of tumor cells and virus-infected cells without prior sensitization and represent unique lymphocytes involved in primary host defense. NKR-P1 is thought to be one of NK receptors mediating activation signals because cross-linking of NKR-P1 activates NK cells to exhibit cytotoxicity and IFN-gamma production. However, molecular mechanism of NK cell activation via NKR-P1 is not well elucidated. In this study, we analyzed the cell surface complex associated with NKR-P1 on NK cells and found that NKR-P1 associates with the FcRgamma chain which is an essential component of Fc receptors for IgG and IgE. The association between FcRgamma and NKR-P1 is independent of Fc receptor complexes. Furthermore, NK cells from FcRgamma-deficient mice did not show cytotoxicity or IFN-gamma production upon NKR-P1 cross-linking. Similarly, NK1.1+ T cells from FcRgamma-deficient mice did not produce IFN-gamma upon NKR-P1 crosslinking. These findings demonstrate that the FcRgamma chain plays an important role in activation of NK cells via the NKR-P1 molecule.

Interferon gamma production by natural killer (NK) cells and NK1.1+ T cells upon NKR-P1 cross-linking.

Natural killer (NK) cells play an important role in immune response by producing interferon gamma (IFN-gamma) as well as exhibiting cytotoxic function. IFN-gamma produced by NK cells has been suggested to be involved in differentiation of T helper cells. On the other hand, the NKR-P1 molecule was recently identified as one of the important NK cell receptors, and it recognizes certain kinds of oligosaccharides on target cells and triggers NK cells for cytotoxicity. In the present study, we found that NK cells produce great amounts of IFN-gamma upon cross-linking of the NKR-P1 molecule. In contrast, stimulation of NK cells with IL-2 induced proliferation without producing IFN-gamma. Similar to NK cells, NK1.1+ T cells also produced IFN-gamma upon NKR-P1 cross-linking. NK1.1+ T cells produced IFN-gamma but not interleukin 4 (IL-4) upon NKR-P1 cross-linking, whereas they secreted both IFN-gamma and IL-4 upon T cell receptor cross-linking. These results indicate that NKR-P1 is a receptor molecule on NK and NK1.1+ T cells that induces not only cytotoxicity but also IFN-gamma production. Our findings provide a new pathway for IFN-gamma production by NK and NK1.1+ T cells through NKR-P1 molecules; it may be essential for immune regulation.

Mitogenic effect of HIV-infected human T cell lines on mouse B cells mediated by surface immunoglobulin.

Following HIV-1 infection, a number of disorders are induced in both normal T and B cells by virus products derived from infected CD4+ T cells. In the present study, we found that HIV-infected, but not uninfected, human T cell lines generated vigorous blastogenesis and proliferation of freshly isolated mouse B cells in a short-term culture. Neither human B cells nor rat B cells showed significant responses to the HIV-infected T cell lines in the present condition. The mitogenic effect of HIV-infected human T cell line requires direct cell-cell interaction between mouse B cells and HIV-infected T cell lines. Since either mitomycin c treatment or paraformaldehyde fixation of HIV-infected T cell lines resulted in complete loss of the mitogenic effect, it seems that de novo synthesized viral products are responsible for this effect. Furthermore, anti-mouse immunoglobulin antibody inhibited completely the B cell stimulation by the HIV-infected human T cell lines. Thus, surface immunoglobulin (sIg) on mouse B cells appears to be an essential molecule which transduces activation signals from HIV-infected human T cells into cytoplasm of the B cells.

Developmental arrest of NK1.1+ T cell antigen receptor (TCR)-alpha/beta+ T cells and expansion of NK1.1+ TCR-gamma/delta+ T cell development in CD3 zeta-deficient mice.

The relationship between the structure of the T cell antigen receptor (TCR)-CD3 complex and development of NK1.1+ T cells was investigated. The TCR complex of freshly isolated NK1.1+ TCR-alpha/beta+ thymocytes contained CD3 zeta homodimers and CD zeta-FcR gamma heterodimers, whereas that of the majority of NK1.1- T cells did not contain FcR gamma. The function of CD3 zeta and FcR gamma in the development of NK1.1+ T cells was determined by analyzing CD3 zeta- and FcR gamma-deficient mice. The NK1.1+ T cells from wild-type and CD3 zeta-deficient mice had equal levels of CD3 expression. However, the development of NK1.1+ TCR-alpha/beta+ T cells was almost completely disrupted in thymus and spleen in CD3 zeta-deficient mice, whereas no alteration was observed in FcR gamma-deficient mice. In contrast, the number of novel NK1.1+ TCR-gamma/delta+ thymocytes expressing a surface phenotype similar to NK1.1+ TCR-alpha/beta+ thymocytes increased approximately six times in CD3 zeta-deficient mice. These findings establish the distinct roles of the CD3 zeta chain in the development of the following different thymic T cell compartments: NK1.1- TCR+, NK1.1+ TCR-alpha/beta+, and NK1.1+ TCR-gamma/delta+ thymocytes, which cannot be replaced by CD3 eta or FcR gamma chains.

Production of minor lymphocyte stimulatory-1a antigens from T cell subsets.

T cell subsets that produce minor lymphocyte stimulatory (Mls) antigens were analyzed using mixed lymphocyte reaction (MLR) in vitro or clonal elimination assay in vivo. When lymph node T cells from B10.BR(Mls-1b) mice were stimulated with various T cell subsets from AKR (Mls-1a) mice in the presence of B10.BR antigen presenting cells (APC), proportions of Mls-1a reactive T cell blasts (V beta 6+, V beta 8.1+) increased. The stimulatory potency of CD8+ T cells was higher than that of CD4+ T cells. Furthermore, among either CD8+ or CD4+ T cell subset, CD44+ T cells appeared to produce larger amounts of Mls-1a antigens than CD44- T cells. More marked difference was demonstrated, when stimulator AKR T cells were being activated by immobilized anti-T cell antigen receptor (TCR) antibody during MLR. Thus, AKR T cells appeared to produce large amounts of Mls-1a antigens on appropriate stimulations. These findings were confirmed by the semiquantitative analysis of mRNA levels of MTV-7 in the AKR T cell subsets. When CD8+CD44+ T cells from (AKR x B10.BR)F1 mice were injected intravenously into [B10.BR-->B10.BR] syngeneic bone marrow (BM) chimeras 1 week after BM reconstitution and proportions of V beta 6+ T cells were quantitated 7 weeks later, significant clonal elimination of V beta 6+ T cells was induced among both thymocyte population and lymph node T cell population in a dose-dependent manner of the inoculated F1 T cells. Inoculation of CD8+CD44-F1 T cells eliminated V beta 6+ T cells less efficiently from lymph node T cells and inoculation of CD4+F1 T cells induced no significant clonal elimination of the V beta 6+ T cells. The present findings demonstrate clearly that CD8+CD44+ T cells represent the cells producing large amounts of Mls-1a antigens and inducing clonal elimination of V beta 6+ T cells in vivo.

Fas-mediated cytotoxicity by freshly isolated natural killer cells.

The expression of Fas ligand on natural killer (NK) cells and Fas-mediated cytotoxicity by NK cells was investigated. Fas ligand mRNA was expressed in freshly isolated NK cells but not in T cells. Furthermore, the Fas ligand was detected on the cell surface of NK cells by staining with soluble Fas molecule. We analyzed the cytolytic activity of NK cells against thymocyte targets from normal and lpr mice, and found that the NK cells killed thymocytes from normal mice but not from lpr mice. On the other hand, splenic T cells did not show any cytotoxicity against either of the thymocyte targets. Similarly, NK cells exhibited cytotoxicity against transfectants expressing Fas antigen but not against parental cells or transfectants expressing a mutant Fas antigen with deleted cytoplasmic region. These results demonstrated that NK cells express Fas ligand and possess the capability of killing target cells expressing Fas antigen on their surface. This finding suggests that NK cells play an important role by eliminating Fas-expressing cells either constitutively or inducibly in peripheral lymphoid organs.

Cytotoxicity of fresh NK1.1+ T cell receptor alpha/beta+ thymocytes against a CD4+8+ thymocyte population associated with intact Fas antigen expression on the target.

Recent studies have revealed that 10-20% of CD4+8- or CD4-8- thymocyte populations contain NK1.1+ T cell receptor (TCR)-alpha/beta+ cells. This subpopulation shows characteristics that are different from NK1.1- CD4+ or NK1.1- CD8+ T cells and seems to have developed in a manner different from NK1.1- T cells. Although extensive studies have been performed on the NK1.1+ TCR-alpha/beta+ thymocytes, the physiological role of the NK1.1+ TCR-alpha/beta+ thymocytes has been totally unclear. In the present study, we found that freshly isolated NK1.1+ TCR-alpha/beta+ thymocytes, but neither whole thymocytes nor lymph node T cells, directly killed CD4+8+ thymocytes from normal syngeneic or allogeneic mice by using a long-term cytotoxic assay in which flow cytometry was used to detect the cytotoxicity. However, only weak cytotoxicity was detected against thymocytes from lpr mice on which the Fas antigen that transduces signals for apoptosis into the cells is not expressed. Furthermore, the NK1.1+ TCR-alpha/beta+ thymocytes exhibited high cytotoxicity against T lymphoma targets transfected with fas genes as compared with the parental T lymphoma targets or target cells transfected with mutated fas genes, which lack the function of transducing signals. On the other hand, NK1.1+ effector thymocytes from gld mice that carry a point mutation in Fas ligand did not kill thymocyte targets from normal mice. The present findings, thus, consistently suggest that the NK1.1+ TCR-alpha/beta+ thymocytes kill a subpopulation among CD4+8+ thymocytes via Fas antigen and in this way regulate generation of T lineage cells in the thymus.

Contribution of host radioresistant T cells to the clonal elimination of minor lymphocyte stimulatory-1a reactive T cells in mouse bone marrow chimeras.

When bone marrow (BM) cells from I-E+ and minor lymphocyte stimulatory (Mls) antigen (Ag) disparate mice (Mls-1b) were transplanted to lethally irradiated Mls-1a mice, Mls-1a reactive T cells were found to be completely deleted from the developing thymocyte population in these [Mls-1b-->Mls-1a] radiation chimeras. It has been shown that BM-derived class II (Ia) positive cells play an essential role in this clonal deletion. Thus, Mls-1a Ag appeared to have been transferred from recipient cells to the Ia+ cells derived from donor BM. These Mls-1a-Ia complexes appear to be responsible for elimination of the Mls-1a reactive T cells that have also been derived from donor BM. However, definition of the cells of the recipient that generate the Mls-1a Ag and transfer them to the BM-derived Ia+ cells has remained unclear to date. In the analysis described herein, we have investigated the tolerogenicity of Mls-1a Ag derived from host T cells which represent a major population of radioresistant cells in the [Mls-1b-->Mls-1a] chimeras. When recipient T cells that had been collected and purified from spleens of [Mls-1b-->Mls-1a] chimeras were administered i.v. into [Mls-1b] chimeras, Mls-1a reactive V beta 6+, V beta 8.1+, or V beta 9+ T cells were completely eliminated. Thus, residual radioresistant host T cells present in the radiation BM chimeras are the cells which produce the Mls-1a Ag. These Mls-1a Ags ultimately contribute to the clonal elimination of Mls-1a reactive T cells from the developing thymocyte population. The present findings indicate that recipient T cells which can survive lethal irradiation and produce intrinsic superantigens alter eventually the T cell repertoire in the thymus which have been developing from precursors of donor BM.

NK1.1+ CD4+ CD8- thymocytes with specific lymphokine secretion.

CD4+8- or CD4-8+ thymocytes have been regarded as direct progenitors of peripheral T cells. However, recently, we have found a novel NK1.1+ subpopulation with skewed T cell antigen receptor (TcR) V beta family among heat-stable antigen negative (HSA-) CD4+8- thymocytes. In the present study, we show that these NK1.1+ CD4+8- thymocytes, which represent a different lineage from the major NK1.1- CD4+8- thymocytes or CD4+ lymph node T cells, vigorously secrete interleukin (IL)-4 and interferon (IFN)-gamma upon stimulation with immobilized anti-TcR-alpha beta antibody. On the other hand, neither NK1.1- CD4+8- thymocytes nor CD4+ lymph node T cells produced substantial amounts of these lymphokines. A similar pattern of lymphokine secretion was observed with the NK1.1+ CD4+T cells obtained from bone marrow. The present findings elucidate the recent observations that HSA- CD4+8- thymocytes secrete a variety of lymphokines including IFN-gamma, IL-4, IL-5 and IL-10 before the CD4+8- thymocytes are exported from thymus. Our evidence indicates that NK1.1+ CD4+8- thymocytes are totally responsible for the specific lymphokine secretions observed in the HSA- CD4+8- thymocytes.

An NK1.1+ CD4+8- single-positive thymocyte subpopulation that expresses a highly skewed T-cell antigen receptor V beta family.

In the present report we describe a CD4+8- heat stable antigen-negative (HSA-) thymocyte subpopulation that expresses a distinguishably low density of alpha beta T-cell antigen receptors (TCRlo) from the majority of CD4+8- high-density TCR (TCRhi) mature-type thymocytes. This subpopulation appears relatively late in life. Analysis of MEL-14, Pgp-1 (CD44), ICAM-1 (CD54), and NK1.1 expression on this subpopulation revealed that the CD4+8- TCRlo population was a population having unique characteristics (MEL-14-, CD44+, ICAM-1+, and NK1.1+) compared to the CD4+8- TCRhi thymocytes, most of which are MEL-14+, CD44-, ICAM-1-, and NK1.1-. When TCR beta-chain variable region (V beta) usage was analyzed, this thymic population expressed predominantly products of V beta 7 and V beta 8.2 TCR gene families. Interestingly, cells with V beta 8.1 TCRs, which are reactive to Mls-1a antigens, were not eliminated from the CD4+8- HSA- TCRlo subpopulation but had been eliminated from the major CD4+8- HSA- TCRhi subpopulation in Mls-1a strains. A subset with a phenotype similar to the CD4+8- HSA- TCRlo thymocytes was also identified primarily in bone marrow, and this subset constituted approximately half of the CD4+ T cells in the bone marrow. The CD4+8- HSA- TCRlo cells showed extremely high proliferative responses to immobilized anti-TCR antibody but generated negligible responses to allogeneic H-2 antigens compared to the responses generated by the major CD4+8- HSA- CD3hi cells. However, the CD4+8- HSA- TCRlo cells in Mls-1b mice mounted vigorous proliferative responses to Mls-1a antigens but not in Mls-1a mice. The properties of this T-cell subset suggest that these cells belong to a lineage distinct from the major T-cell population.

[A study on clonal elimination of auto-reactive thymocytes in bone marrow chimera mice].

Bone marrow (BM) chimera mice were established by injecting BM cells from B10 H-2 congenic or recombinant mice (Mls-1b) into lethally irradiated AKR (Mls-1a) mice in order to elucidate what type of cells were responsible for intrathymic clonal elimination of self-reactive V beta 6+T cells that are reactive to Mls-1a plus I-E products. When I-E+ mice were donors, V beta 6+ SP thymocytes were not eliminated. However, in chimeras where B10 (I-Ab, I-E-) or B10.A(4R)(I-Ak, I-E-) mice were donors, variable proportions of V beta 6+ SP cells were observed. These differences appeared to be attributable to the difference in affinity between class II antigens expressed on BM derived cells and Mls-1a on recipient cells. When AKR mice were reconstituted with BM cells from both B10 and AKR mice, V beta 6+ SP thymocytes were eliminated according to frequencies of the AKR derived cells. These findings collectively indicate that the BM derived thymic stromal cells are essential for the clonal elimination of V beta 6+ cells. However, in GVHR chimeras prepared by injecting of both BM cells and splenic T cells from the same donor mice, V beta 6+ cells were not eliminated at all any periods after BMT. Significantly high number of V beta 6+ SP thymocytes seen 1 week after BMT were shown to be the splenic T cellsinjected with BM cells or their descendants. By contrast, the proportions of the V beta 6+ SP cells in GVHR chimeras 5 weeks after BMT fell within the same range as those of normal donor mice. These V beta 6+ cells were derived from BM precursors. These results reveal that acute GVHR in the thymus results in abrogation of clonal elimination of self reactive T cells in the thymus.

Clonal elimination of self reactive V beta 6+ T cells induced by H-2 products expressed on thymic radio-resistant components.

The role of thymic radio-resistant cells on clonal elimination of V beta 6+ T cells that are reactive to minor lymphocyte stimulatory (Mls)-1a plus I-E antigens has been investigated. Previous studies with allogeneic bone marrow chimeras revealed that radio-sensitive I-E+ cells derived from donor bone marrow in the thymus play a major role in the clonal elimination of V beta 6+ T cells. However, we could show that not only the thymic bone marrow derived components but also the radio-resistant ones (presumably thymic epithelial cells) might be involved in induction of clonal elimination of the self-reactive T cells. The proportion of V beta 6+ T cells present varied with the H-2 haplotype of the thymus and the cell types presenting Mls-1a products, which might be attributable to differences in the affinity of the H-2 products to T cell antigen receptors and differences in the amount of tolerogens expressed on the stromal cells.