NK1.1+ thymocytes. Adult murine CD4-, CD8- thymocytes contain an NK1.1+, CD3+, CD5hi, CD44hi, TCR-V beta 8+ subset.

CD4-, CD8- thymocytes were purified from thymi obtained from normal C57BL/6 mice. By flow cytometry analysis, 5 to 10% of these double negative (DN) thymocytes were found to express NK1.1 on their surface. The NK1.1+ DN thymocytes were demonstrated, by two-color fluorescence, to be CD3lo, CD5hi, CD44hi, J11d-, B220-, MEL 14-, IL2R- with 60% expressing TCR-V beta 8 as determined by the mAb F23.1. In contrast, splenic and peripheral blood NK cells were NK1.1+, CD3-, CD5-, TCR-V beta 8- with 40 to 60% being MEL 14+. Unlike peripheral NK cells, fresh DN thymocytes enriched for NK1.1+ cells were unable to kill YAC-1, the classical murine NK cell target. However, these cells were able to mediate anti-CD3 redirected lysis even when they were assayed immediately after purification, i.e., with no culture or stimulation. These data demonstrate that adult murine thymocytes contain NK1.1+ cells which are distinct, both by function and phenotype, from peripheral NK cells. These data also raise the issue of a possible NK/T bipotential progenitor cell.     

Heat shock protein-GP96 as an innate sensor of damage and activator of autoreactive NKT and regulatory T cells during liver regeneration

Tissue disintegration after injury leads, in the endoplasmic reticulum (ER), to activation of adaptive pathways known as the ER stress response. It is directed to the correction of unfolded proteins and to the activation of proteasome-dependent ER-associated degradation of the misfolded proteins, but induces also a rapid activation of natural and adaptive immunity, since a ER resident heat shock protein-gp96 acts not only as a molecular chaperone, but also as a strong adjuvant, able to cross-present the antigenic peptides onto MHC class I or MHC class II pathways. Analyzing its potential role in processes of normal growth, in mice subjected to 1/3 partial hepatectomy (pHx) we determined the tissue expression of gp96 protein and mRNA in regenerating liver, thymus and spleen, determining simultaneously the phenotypic profile and spontaneous cytotoxic activity of intrahepatic and splenic mononuclear lymphatic cells (MNLC) against NKT- and NK-cells sensitive targets (syngeneic thymocytes and YAC-1) in wild, perforin and FasL deficient mice. The data have shown that pHx induces fast overexpression of gp96 protein and mRNA in hepatocytes, spleen and thymus, with accumulation of CD3intermediate/NK1.1+/CD69+ cells (liver) and Foxp3+CD4+CD25+ cells (liver and thymus). Simultaneously, intrahepatic MNLC acquired the FasL-dependent cytotoxic potential against NKT-sensitive targets and both, intrahepatic and splenic MNLC, acquired the perforin-dependent cytotoxic potential against NK-sensitive targets, implying that during the disturbance of morphostasis gp96 serves as a natural adjuvant for chaperoning antigenic self peptides into the immune surveillance pathways, resulting in activation of autoreactive NKT and regulatory cells, as well as NK cells. Moreover, cell cycle analysis revealed that G2+M phase of regenerating hepatocytes in PKO mice was translocated from the 1st to the 7th p. o. day, as well as that hepatocytes from FasL deficient mice were arrested in G0/G1 phase.     

Lymphokine-activated killer (LAK) cells. VI. NK1.1+, CD3+ LAK effectors are derived from CD4-, CD8-, NK1.1- precursors.

Normal murine splenocytes cultured with IL2 for 6, but not 3, days contained an NK1.1+, CD3+ lytically active subset. These lymphocytes were not derived from NK1.1+ precursors since NK1.1+ cells, purified by flow cytometry, failed to express CD3, as determined by the 145-2C11 mAb, on their surface even after culture with IL2 for 6 days. Instead, the precursors of the NK1.1+, CD3+ effectors were contained in a B cell-depleted CD4-, CD8-, NK1.1- splenic subset. Freshly obtained CD4-, CD8-, NK1.1- splenocytes were mostly CD3+, CD5+, B220-, had no spontaneous lytic activity against YAC-1, and were unable to mediate anti-CD3 directed lysis against FcR-bearing target cells. Culture of the CD4-, CD8-, NK1.1- splenocytes with IL2, for 6 days, resulted in the development of NK1.1+, CD3+, B220+ effectors 40% of which were CD5dim and 20-25% of which expressed TCR-V beta 8 as determined by the F23.1 mAb. The acquisition of NK1.1, B220, and lytic activity by this triple-negative subset was readily inhibited by cyclosporine A (CSA). On the other hand, CSA had no effect on the acquisition of B220 or lytic activity by NK1.1+ precursors obtained by flow cytometry sorting. Moreover, all of the NK1.1+ cells generated by IL2 culture of splenocytes obtained from mice depleted of NK1.1+ lymphocytes (by in vivo injection of anti-NK1.1 mAb) coexpressed CD3 on their surface and were thus distinct from classical NK cells. These findings demonstrate that splenic NK cells do not express or acquire CD3; that the NK1.1+, CD3+ LAK effectors are derived from an NK1.1- precursor; and that CSA is exquisitely selective in its inhibitory effect on LAK generation.     

Natural killer cells in the thymus. Studies in mice with severe combined immune deficiency

The relationship between NK cell and T cell progenitors was investigated by using mice with severe combined immune deficiency (scid). Scid mice are devoid of mature T and B cells because they cannot rearrange their Ig and TCR genes. However, they have normal splenic NK cells. Thymus of scid mice, although markedly hypocellular, contains cells that lyse YAC-1, an NK-sensitive tumor cell. By flow cytometry, two populations of cells were identified in the scid thymus. Eighty percent of the cells were Thy-1+, IL-2R(7D4)+, J11d+, CD3-, CD4-, CD8- whereas the remaining were IL-2R-, J11d-, CD3-, CD4-, and CD8-. By cell sorting, all NK activity was found in the latter population, which is phenotypically similar to splenic NK cells. To determine if the thymus contains a bipotential NK/T progenitor cell, J11d+, IL-2R+ cells were cultured and analyzed for the generation of NK cells in vitro. These cells were used because they resemble 15-day fetal and adult CD4- CD8- thymocytes that are capable of giving rise to mature T cells. Cultured J11d+ thymocytes acquired non-MHC-restricted cytotoxicity, but in contrast to mature NK cells, the resulting cells contained mRNA for the gamma, delta, and epsilon-chains of CD3. This suggests that J11d+ cells are early T cells that can acquire the ability to kill in a non-MHC-restricted manner, but which do not give rise to NK cells in vitro. The differentiative potential of scid thymocytes was also tested in vivo. Unlike bone marrow cells, scid thymocytes containing 80% J11d+ cells failed to give rise to NK cells when transferred into irradiated recipients. Together these results suggest that mature NK cells reside in the thymus of scid mice but are not derived from a common NK/T progenitor.     

Phenotypic and functional heterogeneity of thymic and splenic lymphokine activated killer cells relative to ornithine sensitivity

We have previously reported the selective inhibition of cytotoxic T lymphocytes (CTL) by 10 mM ornithine (ORN) relative to natural killer (NK) cell-derived lymphokine activated killer cells (LAK). To determine if this were due to differences in the progenitor cells or the type of stimulus, we used cortisone-resistant thymocytes (CRT) as a source of mature T cells for induction of LAK and CTL, and compared the results with spleen. Thymic and splenic CTL precursors (CTLp) from C57B1/6 (B6) mice were CD8+, ASGM1-, ORN sensitive. Splenic LAK precursors (LAKp) were CD8-, ASGM1+, ORN resistant when assayed against both YAC-1 and P815 tumor targets. In contrast, CRT-derived LAKp were CD8-, ASGM1+, ORN resistant against YAC-1, whereas LAKp against P815 were CD8+, ASGM1+, ORN sensitive. ORN sensitivity was also observed among CTL and LAK in DBA/2 mice and was associated with CD8+ phenotype. Therefore, our initial observation of differential ORN sensitivity in CTL vs LAK was a function of the progenitor cells; furthermore, CD8+ cytolytic cells are ORN sensitive whether activated by antigen (CTL) or IL-2 (T-LAK).     

Immunological response in the mouse melanoma model after local hyperthermia

Our study was aimed to characterize the phenotype and functional endpoints of local microwave hyperthermia (LHT, 42 degrees C) on tumor infiltrating and spleen leukocytes. The effectiveness of LHT applied into the tumor of B16F10 melanoma-bearing C57/BL6 mice was compared with anesthetized and non-treated animals. Subpopulations of leukocytes were analyzed using the flow cytometry, and the cytotoxic activity of splenocytes against syngeneic B16F10 melanoma and NK-sensitive YAC-1 tumor cell lines was evaluated in (51)Cr-release assay. Similarly, the in vitro modification of the heat treatment was performed using healthy and melanoma-bearing splenocytes. We found a 40 % increase of activated monocytes (CD11b+CD69+) infiltration into the tumor microenvironment. In the spleen of experimental animals, the numbers of cytotoxic T lymphocytes (CTLs-CD3+CD8+) and NK cell (CD49b+NK1.1+) raised by 22 % and 14 %, respectively, while the NK1.1+ monocytes decreases by 37 %. This was accompanied by an enhancement of cytotoxic effector function against B16F10 and YAC-1 targets in both in vivo and in vitro conditions. These results demonstrate that LHT induces better killing of syngeneic melanoma targets. Furthermore, LHT evokes the homing of activated monocytes into the tumor microenvironment and increases the counts of NK cells and CTL in the spleen.     

Regulation of NKT cells by Ly49: analysis of primary NKT cells and generation of NKT cell line

TCRalphabeta(+)NK1.1(+) (NKT) cells are known to express various NK cell-associated molecules including the Ly49 family of receptors for MHC class I, but its functional significance has been unclear. Here, we examined the expression of Ly49A, C/I and G2 on various NKT cell populations from normal and MHC class I-deficient C57BL/6 mice as well as their responsiveness to alpha-galactosylceramide (alpha-GalCer), a potent stimulator of CD1d-restricted NKT cells. The frequency and the level of Ly49 expression varied among NKT cells from different tissues, and were regulated by the expression of MHC class I and CD1d in the host. Stimulation of various NKT cells with alpha-GalCer suggested that Ly49 expression inversely correlates with the responsiveness of NKT cells to alpha-GalCer. Moreover, alpha-GalCer presented by normal dendritic cells stimulated purified Ly49(-), but not Ly49(+), splenic NKT cells, whereas MHC class I-deficient dendritic cells presented alpha-GalCer to both Ly49(+) and Ly49(-) NKT cells equally well. Therefore, MHC class I on APCs seems to inhibit activation of NKT cells expressing Ly49. To further characterize CD1d-restricted NKT cells, we generated an alpha-GalCer-responsive NKT cell line from thymocytes. The line could only be generated from Ly49(-)NK1.1(+)CD4(+) thymocytes but not from other NKT cell subsets, and it lost expression of NK1.1 and CD4 during culture. Together, these results indicate the functional significance of Ly49 expression on NKT cells.     

The influence of CD1d in postselection NKT cell maturation and homeostasis

After being positively selected on CD1d-expressing thymocytes, NKT cells undergo a series of developmental changes that can take place inside or outside the thymus. We asked whether CD1d continues to play a role in late-stage NKT cell development and, in particular, during the functionally significant acquisition of NK1.1 that is indicative of NKT cell maturity. We report that CD1d is indeed crucial for this step, because immature NK1.1(-) NKT cells fail to fully mature when transferred to a CD1d-deficient environment. Surprisingly, however, the lack of CD1d did not greatly affect the long-term survival of NKT cells, and they continued to express CD69 and slowly proliferate. This directly contradicts the currently held view that these phenomena are caused by autoreactivity directed against CD1d/TCR-restricted self-Ags. Our findings demonstrate an ongoing role for TCR-mediated signaling throughout NKT cell development, but the characteristic semiactivated basal state of NKT cells is controlled by CD1d-independent factors or is intrinsic to the cells themselves.     

CD8+ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo

NKT cells express both NK cell-associated markers and TCR. Classically, these NK1.1+TCRalphabeta+ cells have been described as being either CD4+CD8- or CD4-CD8-. Most NKT cells interact with the nonclassical MHC class I molecule CD1 through a largely invariant Valpha14-Jalpha281 TCR chain in conjunction with either a Vbeta2, -7, or -8 TCR chain. In the present study, we describe the presence of significant numbers of NK1.1+TCRalphabeta+ cells within lymphokine-activated killer cell cultures from wild-type C57BL/6, CD1d1-/-, and Jalpha281-/- mice that lack classical NKT cells. Unlike classical NKT cells, 50-60% of these NK1.1+TCRalphabeta+ cells express CD8 and have a diverse TCR Vbeta repertoire. Purified NK1.1-CD8alpha+ T cells from the spleens of B6 mice, upon stimulation with IL-2, IL-4, or IL-15 in vitro, rapidly acquire surface expression of NK1.1. Many NK1.1+CD8+ T cells had also acquired expression of Ly-49 receptors and other NK cell-associated molecules. The acquisition of NK1.1 expression on CD8+ T cells was a particular property of the IL-2Rbeta+ subpopulation of the CD8+ T cells. Efficient NK1.1 expression on CD8+ T cells required Lck but not Fyn. The induction of NK1.1 on CD8+ T cells was not just an in vitro phenomenon as we observed a 5-fold increase of NK1.1+CD8+ T cells in the lungs of influenza virus-infected mice. These data suggest that CD8+ T cells can acquire NK1.1 and other NK cell-associated molecules upon appropriate stimulation in vitro and in vivo.     

Target cell induced activation of NK cells in vitro: cytokine production and enhancement of cytotoxic function

This study examines the effect of fixed AK-5 tumour cells on rat NK cells. Co-culture of NK cells with fixed tumour cells augmented the cytotoxicity of NK cells against NK-sensitive targets, YAC-1 and AK-5, and induced the secretion of IFN-gamma by NK cells. Antibody against IFN-gamma suppressed the anti-tumour activity of NK cells, whereas the addition of T cells during co-culture enhanced this activity. However, macrophages and B cells had no significant effect when present during co-culture with NK cells. All the inducible cytotoxicity was contained within the NK (CD161+) and NKT (CD3+, CD161+) subsets of lymphocytes. However, in the presence of T cells, the cytolytic potential of NKT cells was higher than that of NK cells alone. The augmentation of cytotoxic activity of NK cells by AK-5 cells in presence of T cells was dependent on IL-2 and IFN-gamma secretion. NK cell activation was blocked by specific antibodies to IL-2 and IFN-gamma in the presence of T cells. Interaction between fixed AK-5 cells with NK and T cell populations induced the expression of Fas-L and perforin in NK cells. These data demonstrate that fixed AK-5 cells initiated cytokine synthesis by NK cells, and the enhanced cytotoxic activity in the presence of T cells was induced as a consequence of the products secreted by activated T lymphocytes. The present observations reflect the possible interactions taking place in vivo after the transplantation of AK-5 tumour in animals. They also suggest direct activation of NK cells after their interaction with the tumour cells.     

Control of NKT cell differentiation by tissue-specific microenvironments

CD1d-restricted Valpha14 NKT cells play an important role in both Th1- and Th2-type immune responses. To determine whether NKT cells develop two functionally distinct subsets that provoke different types of responses, we examined the phenotypes and cellular functions of NK1.1(+) and DX5(+) T cells. We found that both NK1.1(+) and DX5(+) T cells are CD1d-restricted Valpha14 T cells with identical Ag specificities, phenotypes, tissue locations, and functions. Similar to the NK1.1 marker, the DX5 marker (CD49b) is expressed on mature NKT cells in both NK1.1 allele-positive and allele-negative strains. However, when NK1.1(+) and DX5(+) NKT cells isolated from different tissues were compared, we found that thymic and splenic NKT cells differed not only in their cytokine profiles, but also in their phenotype and requirements for costimulatory signals. Thymic NKT cells displayed the phenotype of activated T cells and could be fully activated by TCR ligation. In contrast, splenic NKT cells displayed the phenotype of memory T cells and required a costimulatory signal for activation. Furthermore, the function and phenotype of thymic and splenic NKT cells were modulated by APCs from various tissues that expressed different levels of costimulatory molecules. Modulation of NKT cell function and differentiation may be mediated by synergic effects of costimulatory molecules on the surface of APCs. The results of the present study suggest that the costimulatory signals of tissue-specific APCs are key factors for NKT cell differentiation, and these signals cannot be replaced by anti-CD28 or anti-CD40 ligand Abs.     

IL-15 expands unconventional CD8alphaalphaNK1.1+ T cells but not Valpha14Jalpha18+ NKT cells

Despite recent gains in knowledge regarding CD1d-restricted NKT cells, very little is understood of non-CD1d-restricted NKT cells such as CD8(+)NK1.1(+) T cells, in part because of the very small proportion of these cells in the periphery. In this study we took advantage of the high number of CD8(+)NK1.1(+) T cells in IL-15-transgenic mice to characterize this T cell population. In the IL-15-transgenic mice, the absolute number of CD1d-tetramer(+) NKT cells did not increase, although IL-15 has been shown to play a critical role in the development and expansion of these cells. The CD8(+)NK1.1(+) T cells in the IL-15-transgenic mice did not react with CD1d-tetramer. Approximately 50% of CD8(+)NK1.1(+) T cells were CD8alphaalpha. In contrast to CD4(+)NK1.1(+) T cells, which were mostly CD1d-restricted NKT cells and of which approximately 70% were CD69(+)CD44(+), approximately 70% of CD8(+)NK1.1(+) T cells were CD69(-)CD44(+). We could also expand similar CD8alphaalphaNK1.1(+) T cells but not CD4(+) NKT cells from CD8alpha(+)beta(-) bone marrow cells cultured ex vivo with IL-15. These results indicate that the increased CD8alphaalphaNK1.1(+) T cells are not activated conventional CD8(+) T cells and do not arise from conventional CD8alphabeta precursors. CD8alphaalphaNK1.1(+) T cells produced very large amounts of IFN-gamma and degranulated upon TCR activation. These results suggest that high levels of IL-15 induce expansion or differentiation of a novel NK1.1(+) T cell subset, CD8alphaalphaNK1.1(+) T cells, and that IL-15-transgenic mice may be a useful resource for studying the functional relevance of CD8(+)NK1.1(+) T cells.     

Cutting edge: contribution of NK cells to the homing of thymic CD4+NKT cells to the liver

In contrast to peripheral lymphoid organs, in the liver a high proportion of T cells are CD4+NKT cells. We have previously reported that LFA-1 plays a pivotal role in the homing of thymic CD4+NKT cells to the liver. In the present study, we further assessed which cell type participates in the homing of thymic CD4+NKT cells to the liver. The accumulation of donor thymocyte-derived CD4+NKT cells in the liver of SCID mice that had been reconstituted with thymocytes from C57BL/6 mice was severely impaired by in vivo depletion of NK cells, but not Kupffer cells in recipients. These results suggest that NK cells participate in the homing of thymic CD4+NKT cells to the liver. We assume that LFA-1 expressed on NK cells is involved in this mechanism.     

Induction of CD4+ murine natural killer T-like cells by immunization with syngeneic thymoma expressing embryonic alpha-fetoprotein

In embryo, before the establishment of acquired immunity, a variety of embryonic antigens like alpha-fetoprotein (AFP) are produced and secreted in the sera, which rapidly disappear after the birth. Such embryonic antigens sometimes reappear from various tumor cells and decrease in the case of remission, indicating embryonic antigens may alert immune system to control tumors. In the present study, to examine the evoked immune responses against the tumors expressing embryonic antigen, we administered AFP-gene-transfected EL4 cells into syngeneic C57BL/6 mice and established a killer line against the tumor cells. To our surprise, the killer line was CD4+ NK1.1+, natural killer T (NKT)-like cells and eliminated not only AFP-expressing EL4 but YAC-1 cells. Moreover, the established line uniformly expressed Vbeta11 and secreted IL-4, IL-10, IL-13, and IFN-gamma. In vivo inoculation of the line markedly reduced the tumor growth in SCID mice, suggesting novelty of the NKT-like line for tumor surveillance.     

Targeted expression of human CD1d in transgenic mice reveals independent roles for thymocytes and thymic APCs in positive and negative selection of Valpha14i NKT cells

CD1d-dependent invariant Valpha14 (Valpha14i) NKT cells are innate T lymphocytes expressing a conserved semi-invariant TCR, consisting, in mice, of the invariant Valpha14-Jalpha18 TCR alpha-chain paired mostly with Vbeta8.2 and Vbeta7. The cellular requirements for thymic positive and negative selection of Valpha14i NKT cells are only partially understood. Therefore, we generated transgenic mice expressing human CD1d (hCD1d) either on thymocytes, mainly CD4+ CD8+ double positive, or on APCs, the cells implicated in the selection of Valpha14i NKT cells. In the absence of the endogenous mouse CD1d (mCD1d), the expression of hCD1d on thymocytes, but not on APCs, was sufficient to select Valpha14i NKT cells that proved functional when activated ex vivo with the Ag alpha-galactosyl ceramide. Valpha14i NKT cells selected by hCD1d on thymocytes, however, attained lower numbers than in control mice and expressed essentially Vbeta8.2. The low number of Vbeta8.2+ Valpha14i NKT cells selected by hCD1d on thymocytes was not reversed by the concomitant expression of mCD1d, which, instead, restored the development of Vbeta7+ Valpha14i NKT cells. Vbeta8.2+, but not Vbeta7+, NKT cell development was impaired in mice expressing both hCD1d on APCs and mCD1d. Taken together, our data reveal that selective CD1d expression by thymocytes is sufficient for positive selection of functional Valpha14i NKT cells and that both thymocytes and APCs may independently mediate negative selection.     

NK cell responses to Plasmodium infection and control of intrahepatic parasite development

Various components of innate and adaptive immunity contribute to host defenses against Plasmodium infection. We investigated the contribution of NK cells to the immune response to primary infection with Plasmodium yoelii sporozoites in C57BL/6 mice. We found that hepatic and splenic NK cells were activated during infection and displayed different phenotypic and functional properties. The number of hepatic NK cells increased whereas the number of splenic NK cells decreased. Expression of the Ly49 repertoire was modified in the spleen but not in the liver. Splenic and hepatic NK cells have a different inflammatory cytokines profile production. In addition, liver NK cells were cytotoxic to YAC-1 cells and P. yoelii liver stages in vitro but not to erythrocytic stages. No such activity was observed with splenic NK cells from infected mice. These in vitro results were confirmed by the in vivo observation that Rag2(-/-) mice were more resistant to sporozoite infection than Rag2(-/-) gamma c(-/-) mice, whereas survival rates were similar for the two strains following blood-stage infection. Thus, NK cells are involved in early immune mechanisms controlling Plasmodium infection, mostly at the pre-erythrocytic stage.     

Liver CD4-CD8- NK1.1+ TCR alpha beta intermediate cells increase during experimental malaria infection and are able to exhibit inhibitory activity against the parasite liver stage in vitro

Experimental infection of C57BL/6 mice by Plasmodium yoelii sporozoites induced an increase of CD4-CD8- NK1.1+ TCR alpha beta int cells and a down-regulation of CD4+ NK1.1+ TCR alpha beta int cells in the liver during the acute phase of the infection. These cells showed an activated CD69+, CD122+, CD44high, and CD62Lhigh surface phenotype. Analysis of the expressed TCRV beta segment repertoire revealed that most of the expanded CD4-CD8- (double-negative) T cells presented a skewed TCRV beta repertoire and preferentially used V beta 2 and V beta 7 rather than V beta 8. To get an insight into the function of expanded NK1.1+ T cells, experiments were designed in vitro to study their activity against P. yoelii liver stage development. P. yoelii-primed CD3+ NK1.1+ intrahepatic lymphocytes inhibited parasite growth within the hepatocyte. The antiplasmodial effector function of the parasite-induced NK1.1+ liver T cells was almost totally reversed with an anti-CD3 Ab. Moreover, IFN-gamma was in part involved in this antiparasite activity. These results suggest that up-regulation of CD4-CD8- NK1.1+ alpha beta T cells and down-regulation of CD4+ NK1.1+ TCR alpha beta int cells may contribute to the early immune response induced by the Plasmodium during the prime infection.     

Murine granulated metrial gland cells at uterine implantation sites are natural killer lineage cells

Granulated metrial gland (GMG) cells, a population of morphologically distinct, bone marrow-derived cells in murine decidua that react with mAb 4H12, are shown in this report to express NK-specific Ag and to become cytolytic to the NK cell target YAC-1 when cultured in the lymphokine IL-2. When 1-mm3 explants of 8-day decidual tissue were cultured with IL-2, large numbers of 4H12+ GMG cells migrated out of the tissue. Migration was dependent on the amount of IL-2 used. This explant technique was used to isolate a pure population of GMG cells. The migratory activated GMG cells were phenotypically 4H12+, NK1.1+, LGL-1+/-, CD3-, and MAC-1-. Furthermore, the IL-2-activated GMG cells killed YAC-1 but not P815 cells in a 4-h 51Cr-release cytotoxicity assay. 4H12+ GMG cells from collagenase-digested decidual tissue also were analyzed for the presence of NK lineage Ag by flow cytometry and shown to coexpress the NK-associated Ag NK1.1 and ASGM1 but not the T cell Ag CD3 or macrophage Ag MAC-1 or F4/80. GMG cells isolated by collagenase digestion did not express LGL-1, an Ag associated with lytic NK cells. Our results demonstrate that GMG cells express Ag and functions characteristic of NK cells, and thus GMG cells can be assigned to the NK lineage. The possible relevance of NK cells at implantation sites is discussed.     

Alpha-glycosylceramides enhance the antitumor cytotoxicity of hepatic lymphocytes obtained from cancer patients by activating CD3-CD56+ NK cells in vitro

Alpha-glycosylceramides, such as alpha-galactosylceramide and alpha-glucosylceramide, induce antitumor immunity in various murine cancer models. In the murine hepatic metastasis model, V alpha 14 TCR+NK1.1+ T cells, which accumulate preferentially in the liver, are considered to play a key role in the induction of antitumor immunity by alpha-glycosylceramides. We recently reported that V alpha 24 TCR+ NKT cells, the human homologues of murine V alpha 14 TCR+NK1.1+ cells, are rarely seen among freshly isolated human hepatic lymphocytes. Therefore, it is important to examine whether alpha-glycosylceramides also enhance the antitumor cytotoxicity of human hepatic lymphocytes, as they have been shown to do in murine systems, to determine the usefulness of alpha-glycosylceramides in cancer immunotherapy in humans. Here, we show that alpha-glycosylceramides greatly enhance the cytotoxicity of human hepatic lymphocytes obtained from cancer patients against the tumor cell lines, K562 and Colo201, in vitro. The direct effector cells of the elicited cytotoxicity were CD3-CD56+ NK cells. Even though V alpha 24 TCR+NKT cells proliferated remarkably in response to alpha-glycosylceramides, they did not contribute directly to the cytotoxicity. Our observations strongly suggest the potential usefulness of alpha-glycosylceramides for immunotherapy of liver cancer in humans based on their ability to activate CD3-CD56+ NK cells in the liver.