Three day neonatal thymectomy selectively depletes NK1.1+ T cells

Neonatal thymectomy of mice 3 days after birth but not at birth leads to T cell-mediated, organ-specific, autoimmune disease in a strain-dependent manner. The mechanisms that lead to disease in this model remain unknown, but the answer may lie in a deficiency of thymus-dependent cells or factors. One candidate is the relatively rare population of NK1.1 + T cells (NKT cells). Conventional alphabetaTCR+ T cells appear in the thymus from days 17-18 of embryogenesis and start emigrating to the periphery around birth, whereas the development of NKT cells is thought to be delayed until at least 1 week after birth. We have confirmed this to be the case in both (BALB/c x C57BL/6)F1 (autoimmune susceptible) and C57BL/6 (autoimmune resistant) mice. Moreover, examination of T cells (in spleen, lymph nodes, liver and bone marrow) from mice following 3 day neonatal thymectomy revealed a significant reduction in the presence of NKT cells in all tissues. However, the extent of depletion was generally more pronounced in (BALB/c x C57BL/6)F1 than in C57BL/6 mice, and the few remaining NKT cells in C57BL/6 mice were enriched for a CD4-CD8int subset which is absent from the thymus and may represent a distinct lineage of thymus-independent NKT cells. Given mounting evidence of a role for NKT cells in protection from autoimmune disease, it is possible that their specific removal by neonatal thymectomy may contribute to the susceptibility of these mice to autoimmune disease.     

Role of NK1.1+ T cells in a TH2 response and in immunoglobulin E production

Immune responses dominated by interleukin-4 (IL-4)-producing T helper type 2 (TH2) cells or by interferon gamma (IFN-gamma)-producing T helper type 1 (TH1) cells express distinctive protection against infection with different pathogens. Interleukin-4 promotes the differentiation of na?ve CD4+ T cells into IL-4 producers and suppresses their development into IFN-gamma producers. CD1-specific splenic CD4+NK1.1+ T cells, a numerically minor population, produced IL-4 promptly on in vivo stimulation. This T cell population was essential for the induction of IL-4-producing cells and for switching to immunoglobulin E, an IL-4-dependent event, in response to injection of antibodies to immunoglobulin D.     

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.     

Impaired NK1.1+ T cells do not prevent the development of an IgE-dependent allergic phenotype

BACKGROUND: The induction of TH2 immune responses is critically dependent on initial IL-4. Although crucial, the source of this early IL-4 has not been identified. One candidate is a CD1 restricted NK1.1+ T cell subpopulation which is known to produce such early IL-4. OBJECTIVES AND METHODS: The necessity of NK1.1+ T cells for the expression of an IgE-dependent phenotype was investigated in a NK1.1+ T cell deficient mouse model. The allergic phenotype was defined as immediate cutaneous hypersensitivity. It was induced by immunization of mice with ovalbumin. Mouse strains used were C57BL/6 mice and C57BL/6 mice homozygous for a targeted mutation of the beta2 microglobulin gene with consecutive loss of CD1 expression, which leads to a drastic reduction of NK1.1+ T cells. Manifestation of an allergic sensitization was assessed by intradermal allergen challenge after i.v. injection of Evans blue solution. The blue stained weal formations were quantified with the Bonitur method. In addition, the Th2 response was confirmed by the measurement of cytokines and serum immunoglobulins. The capability to produce early IL-4 was tested through the assessment of IL-4 mRNA shortly after a single challenge. RESULTS: Wild type and mutated mice did not differ in any of the immunological parameters measured. CONCLUSION: A single exposure to antigen with or without adjuvant induces early IL-4 production in C57BL/6 beta2m-/- mice. This early IL-4 is therefore independent of the presence of NK1.1+ T cells and functional MHC class I molecules and leads to IgE production and immediate cutaneous hypersensitivity.     

MHC class II-dependent NK1.1+ gammadelta T cells are induced in mice by Salmonella infection

We observed the emergence of a novel population of gammadelta T cells expressing NK1.1 Ag in the peritoneal cavity of mice infected with Salmonella choleraesuis. The NK1.1+gammadelta T cells accounted for approximately 20% of all gammadelta T cells emerging in the peritoneal cavity of C57BL/6 mice and expressed preferentially rearranged Vgamma4-Jgamma1 and Vdelta6.3-Ddelta1-Ddelta2-Jdelta1 genes with N diversity. The gammadelta T cells proliferated vigorously in response to PHA-treated spleen cells and produced IFN-gamma in the culture supernatant. However, spleen cells from Abetab-deficient mice were unable to stimulate the gammadelta T cells. Furthermore, the NK1.1+gammadelta T cells were stimulated not only by Chinese hamster ovary (CHO) cells expressing wild-type IAb but also by those expressing IAb/Ealpha52-68 or IAb/pigeon cytochrome c-derived analogue peptide complex. These proliferation activities were inhibited by mAb specific for IAb chain. Consistent with these findings, the emergence of NK1.1+gammadelta T cells was reduced in the peritoneal cavity of Abetab-deficient mice after Salmonella infection, whereas NK1.1+gammadelta T cells were rather abundant in the peritoneal cavity of Salmonella-infected beta2m-deficient mice. Moreover, the NK1.1+gammadelta T cells were easily identified in the thymus of beta2m-deficient but not Abetab-deficient mice. Our results indicated that MHC class II expression is essential for development and activation of NK1. 1+gammadelta T cells in the thymus and the periphery.     

Developmentally regulated extinction of Ly-49 receptor expression permits maturation and selection of NK1.1+ T cells

Clonally distributed inhibitory receptors negatively regulate natural killer (NK) cell function via specific interactions with allelic forms of major histocompatibility complex (MHC) class I molecules. In the mouse, the Ly-49 family of inhibitory receptors is found not only on NK cells but also on a minor (NK1.1+) T cell subset. Using Ly-49 transgenic mice, we show here that the development of NK1.1+ T cells, in contrast to NK or conventional T cells, is impaired when their Ly-49 receptors engage self-MHC class I molecules. Impaired NK1.1+ T cell development in transgenic mice is associated with a failure to select the appropriate CD1-reactive T cell receptor repertoire. In normal mice, NK1.1+ T cell maturation is accompanied by extinction of Ly-49 receptor expression. Collectively, our data imply that developmentally regulated extinction of inhibitory MHC-specific receptors is required for normal NK1.1+ T cell maturation and selection.     

Xenospecific CD8+ cytotoxic T lymphocyte generation: accessory function for CD4+ T cells and natural killer 1.1+ cells

BACKGROUND: Controversy exists as to whether natural killer (NK)1.1+ cells additionally support cytotoxic T lymphocyte (CTL) generation. We have previously demonstrated that mice generate a strong in vitro xenospecific CTL response in local popliteal lymph nodes (LN) to footpad immunizations with large numbers of human tumor cells. METHODS: In vivo depletion of various LN subsets using cytotoxic monoclonal antibodies was used to determine their relative importance in stimulating xenospecific CD8+ CTL responses to human Jurkat tumor cells. Depletion of functional NK cells in vivo was evidenced by the relative lack of NK1.1+ cells and NK activity in the spleens and LN of anti-NK1.1 monoclonal antibody-treated mice. CONCLUSION: Depletion of LN subsets indicated that CD4+ T cells were critical in generating an effective xenospecific CD8+ CTL response, but also suggested that NK1.1+ cells play a significant additional accessory role in the development of mouse anti-human xenospecific CTL.     

Involvement of both donor cytotoxic T lymphocytes and host NK1.1+ T cells in the thymic atrophy of mice suffering from acute graft-versus-host disease

The control and eventual eradication of H5N2 influenza virus from domestic poultry in Mexico is dependent on the use of avian influenza (AI) vaccine strategies. This study was performed to determine the amount of hemagglutinin (HA) antigen required to control the signs of disease from a highly pathogenic H5N2 influenza virus (A/Chicken/ Queretaro/19/95) and the amount of antigen required to prevent shedding of virus from vaccinated birds. Six commercial inactivated water in oil H5N2 vaccines available in Mexico were compared with standardized vaccines to assess their efficacy. The amount of HA required to prevent the signs of disease from A/Chicken/Queretaro/19/95 influenza virus was approximately 0.4 microgram per dose. Each of the six commercially available vaccines prevented disease signs, and half of the vaccines significantly reduced viral shedding from vaccinated birds. There is a need for standardization of AI virus vaccine, and the antigen content should be increased in some of the commercially available AI vaccines in Mexico.     

Participation of NK1.1+ T cells in the rejection of lpr alphabetaT cells when bone marrow cells of lpr mice are transplanted into B6 mice

BACKGROUND: Cause-of-death statistics are widely used for comparing health characteristics of European Community (EC) countries. Before attempting to interpret between-country differences, it is essential to assess the biases affecting the comparability of the data. EUROSTAT decided to address globally this problem with the objective to improve the quality and comparability of cause-of-death data within the EC. METHODS: The material is based on a review of results of international comparative cause-of-death studies and on specific inquiries among EC. Both cause-of-death certification and codification practices are analysed. Certification is studied comparing the models of death certificates, the type of information captured, certifiers training and querying practices. The different coding systems are analysed (International classification of diseases (ICD) in use, interpretation of the ICD rules, implementation of automated coding systems). RESULTS: International studies on comparability of certification and coding practices between countries are rare. These studies are based on certification of cases histories and recoding of samples of death certificates. Recent studies on respiratory diseases, cancers and diabetes outline differences that influenced on the reported level of mortality. The specific EUROSTAT investigation (1997) outline general discrepancies: models of death certificates, nature and amount of information entered, way to establish the diagnosis, degree of consistency of the certification process, autopsy practices, certifiers practices, implementation of ICD-10 and implementation of automated coding systems. CONCLUSION: EUROSTAT studies are now focused on causes of death requiring special attention for comparability (e.g. suicide, accidental deaths, drug and alcohol related deaths, unknown and ill-defined causes), on procedures to improve the homogeneity of certifiers training and querying practices, on the effect of the transition to ICD-10. The international model of death certificate recommended by the World Health Organization should be adopted as widely as possible. Uniform complementary information (e.g. surgery, pregnancy, autopsy, place of occurrence of accidental deaths, work accident) should also be adopted. The EUROSTAT investigations must result in definitions of common recommendations and guidelines to EC.     

Immunosuppressive activity of cloned natural killer (NK1.1+) T cells established from murine tumor-infiltrating lymphocytes

To elucidate the role of NK1.1+ T cells in the antitumor immune response, we established cloned NK1.1+ T cell lines from tumor-infiltrating lymphocytes (TIL) of B16 melanoma, and examined their mode of action in generating antitumor effector T cells both in vitro and in vivo. An NK1.1+ T cell clone (TM4.2) was phenotypically CD3+ TCR-alphabeta+ CD4- CD8- NK1.1+, and CD28+. The TM4.2 cells suppressed the in vitro generation of anti-B16 melanoma CTLs, but not the effector function of CTLs. The results using a transwell membrane suggested that their suppressive activity was mediated by both soluble factors and a direct cell to cell interaction. As for the soluble factors, the suppressive activity of the culture supernatant of TM4.2 cells was neutralized by anti-TGF-beta mAb, and the TM4.2 cells actually produced a considerable amount of TGF-beta. On the other hand, the TM4.2 cells showed a high level of cytolytic activity against B cell blasts and CD80-transfected P815, and such cytolytic activity was reduced by the addition of anti-CD80 mAb. In addition, NK1.1+ T cells in the freshly isolated TIL were revealed to express CD28. Furthermore, the TM4.2 cells suppressed the in vitro generation of anti-allo CTLs irrespective of the MHC haplotype. Finally, the TM4.2 cells suppressed the in vivo antitumor immune response. Collectively, these findings demonstrate that NK1.1+ T cells in TIL show immunosuppressive activity in the antitumor immune response through the production of TGF-beta and the preferential cytolysis of B7-expressing cells.     

Effect of macrophage colony-stimulating factor on mouse NK 1.1+ cell activity in vivo

The effect of recombinant human macrophage colony-stimulating factor (rhM-CSF) on NK 1.1+ cell activity in vivo and in vitro was studied. An intravenous injection of rhM-CSF increased the numbers of NK 1.1+ cells in mouse spleen and blood and augmented the clearance of Yac-1 cells in vivo. Using a magnetic cell sorter (MACS), we purified NK 1.1+ cells from vehicle-injected and rhM-CSF-injected mouse spleen cells. More than 95% of the collected cells were NK 1.1 antigen-positive. NK 1.1+ cells purified from rhM-CSF-injected mouse spleen cells exhibited (a) higher cytotoxic activity against Yac-1 cells, (b) higher proliferative responsiveness to interleukin (IL)-2 and (c) a greater production of interferon (IFN)-gamma in response to IL-2 and IL-12 compared to cells purified from vehicle-injected mouse spleen cells in vitro. These results suggest that the administration of rhM-CSF increases NK 1.1+ cell numbers and activates the cells in vivo.     

IL-2 and IL-7 differentially induce CD4-CD8- alpha beta TCR+NK1.1+ large granular lymphocytes and IL-4-producing cells from CD4-CD8- alpha beta TCR+NK1.1- cells: implications for the regulation of Th1- and Th2-type responses

Effector functions of CD4-CD8- double negative (DN) alpha beta TCR+ cells were examined. Among mouse DN alpha beta TCR+ thymocytes, NK1.1+ cells expressing a canonical V alpha 14/J alpha 281 TCR but not NK1.1- cells produce IL-4 upon TCR cross-linking and IFN-gamma upon cross-linking of NK1.1 as well as TCR. Production of IL-4 but not IFN-gamma from DN alpha beta TCR+NK1.1+ cells was markedly suppressed by IL-2. Whereas V alpha 14/J alpha 281 TCR+ cells express NK1.1+, these cells are not the precursor of DN alpha beta TCR+NK1.1+CD16+B220+ large granular lymphocytes (LGL). IL-2 induces rapid proliferation and generation of NK1.1+ LGL from DN alpha beta TCR+NK1.1- but not from DN alpha beta TCR+NK1.1+ cells. LGL cells exhibit NK activity and produce IFN-gamma but not IL-4 upon cross-linking of surface TCR or NK1.1 molecules. In contrast to IL-2, IL-7 does not induce LGL cells or NK activity from DN alpha beta TCR+NK1.1- cells but induces the ability to produce high levels of IL-4 upon TCR cross-linking. Our results show that DN alpha beta TCR+ T cells have several distinct subpopulations, and that IL-2 and IL-7 differentially regulate the functions of DN alpha beta TCR+ T cells by inducing different types of effector cells.     

Fetal liver contains committed NK progenitors, but is not a site for development of CD34+ cells into T cells

The presence of T and NK cells in the human fetal liver and the fact that fetal liver hemopoietic progenitor cells develop into T and NK cells suggest a role for the fetal liver compartment in T and NK cell development. In this work, we show that the capacity of fetal liver progenitors to develop into T cells, in a human/mouse fetal thymic organ culture system, is restricted to an immature subset of CD34+ CD38- cells. No T cell-committed precursors are contained within the more differentiated CD34+ CD38+ population. This conclusion is supported by the observations that no TCR-delta gene rearrangements and no pre-TCR-alpha expression can be detected in this population. However, NK cells were derived from CD34+ CD38- and CD34+ CD38+ fetal liver cells cultured in the presence of IL-15, IL-7, and Flt-3 ligand. Eighty to ninety percent of cells arising from the CD34+ CD38+ population expressed the NK cell-associated markers CD56, CD16, CD94, and NKR-P1A. Several subpopulations of NK cell precursors were identified by differential expression of these receptors. Based on the detection of populations with a similar antigenic profile in freshly isolated fetal liver cells, we propose a model of NK cell differentiation. Collectively, our findings suggest that CD34+ cells differentiate into NK cells, but not into mature T cells, in the human fetal liver.     

Phenotypes and invariant alpha beta TCR expression of peripheral V alpha 14+ NK T cells

A novel subset of peripheral T cells, peripheral NK T cells, is found to be a major population comprising 5% of splenic T and 40% of bone marrow T cells. The majority of peripheral NK T cells are characterized by the expression of an invariant TCR-alpha encoded by V alpha 14/J alpha 281 with a one nucleotide N region. Moreover, a specific reduction of V alpha 14+ NK T cells has been demonstrated to be tightly associated with various autoimmune diseases, indicating their decisive role in autoimmune disease development. In this study, we investigated the phenotypes of peripheral V alpha 14+ NK T cells and their TCR-beta repertoire. Peripheral V alpha 14+ NK T cells, comprise two populations, i.e., small and large sized cells, at an equal frequency, belonged to the CD4- CD8- fraction, and are heat stable antigen(bright), macrophage-1bright, B220bright, CD45RBdim, and Mel-14dim, but CD5-, distinct from thymic NK T cells. TCR-beta analysis clearly showed that peripheral V alpha 14+ NK T cells utilized two to three dominant invariant TCR-beta, such as V beta 8.2 D beta J beta 2.5/V beta 7 D beta J beta 2.1 in the spleen and liver, V beta 8.2 D beta J beta 2.5/V beta 8.3 D beta J beta 2.2/V beta 7 D beta J beta 2.6 in the bone marrow, and V beta 7 D beta J beta 2.1/V beta 3 D beta J beta 1.2 in intestinal intraepithelial lymphocytes. Judging from the unusual surface phenotypes, such as heat stable antigen, macrophage-1, B220, CD45RBdim, and Mel-14dim, which are known to be T cell activation markers, peripheral V alpha 14+ NK T cells may always be activated under physiologic conditions, resulting in the oligoclonal expansion of V alpha 14+ NK T cells with different invariant TCR-beta in different peripheral organs. The unique features of V alpha 14+ NK T cells are discussed.     

Requirement of IL-4 and liver NK1+ T cells for concanavalin A-induced hepatic injury in mice

Con A-induced hepatic injury of mice accompanied by elevated transaminase was inhibited after in vivo depletion of liver NK cells and NK1+ T cells with intermediate TCR by anti-NK1 Ab or anti-IL-2Rbeta Ab. However, depletion of liver NK cells alone by anti-asialo-GM1 Ab did not inhibit hepatic injury. Although depletion of NK1+ T cells inhibited Con A-induced IL-2R expression of CD4+ high TCR (TCRhigh) cells and IL-4 mRNA expression of hepatic mononuclear cells, exogenous IL-4 engendered Con A-induced hepatic injury and endowed the expression of IL-2R of CD4+ TCRhigh cells. It was also found that in vivo treatment with anti-IL-4 Ab before Con A administration inhibited Con A-induced hepatic injury. In addition, although Con A did not induce hepatic injury in MHC class I-deficient mice, exogenous IL-4 again engendered severe hepatic injury in these mice. Further, while serum TNF-alpha levels induced by Con A were greatly decreased in NK1+ T cell-depleted mice and class I-deficient mice, TNF-alpha levels were recovered by exogenous IL-4. These findings reveal that although CD4+ TCRhigh cells in the liver and their production of TNF-alpha are the direct effectors of Con A-induced hepatic injury, liver NK1+ T cells also play an important role in this hepatitis model. Con A hepatitis may serve as an experimental model for human autoimmune hepatitis.     

CD4+ T cells orchestrate both amplification and deletion of CD8+ T cells

This review focuses on the role of CD4+ T cells in regulating immune responses, orchestrating both the amplification and deletion of immune cells, particularly CD8+ T cells. These two functions, which represent only an apparent contradiction, appear to be two faces of the same process of regulation. In fact, because the immune response, once activated, needs to be carefully controlled or switched off when the antigenic stimulus is eliminated, the immune system has developed several strategies either to regulate clonal amplification or to avoid useless expansion of activated cells. In particular, we have reported many data demonstrating that CD4+ T cells may be indicated as the regulatory element in the activation as well as the deletion of CD8+ T cells. New data are also reported on the ability of anergic CD4+ T cells to suppress CD8+ T-cell activation through induction of apoptosis, and on the need for CD8+ T cells for antigen recognition in inducing cell death in CD4+ T cells. Moreover, the central role of CD4+ T cells in the maintenance of peripheral tolerance has been widely described.     

Cutting edge: CD4+ T cells kill CD8+ T cells via Fas/Fas ligand-mediated apoptosis

To explore the possibility that CD4+ T cells, described to mediate the elimination of themselves or B lymphocytes, could also mediate the elimination of CD8+ T cells, we analyzed apoptotic phenomena in cocultures of CD4+ and CD8+ autologous T cell lines. The data show that CD8+ T cells were lysed by activated CD4+ helper T cells by a Fas/FasL-mediated mechanism. CD4+ T cells were not lysed by activated CD8+ T cells, although Fas and FasL were equally expressed and anti-Fas Abs induced apoptosis in both CD4+ and CD8+ T cell populations. The results allowed us to speculate that CD4+ T cells not only help CD8+ T lymphocytes to mature into effector killer cells and to sustain this function but can also limit their growth.     

Antimetastatic effect of NK1+ T cells on experimental haematogenous tumour metastases in the liver and lungs of mice

Depletion of both natural killer 1.1+ (NK1+) intermediate alpha beta T-cell receptor (int T) cells and NK cells by in vivo treatment with anti-NK1 antibody greatly increased hepatic metastases of intravenously injected EL4 cells as well as pulmonary metastases of 3LL cells in C57BL/6 mice. However, depletion of NK cells alone by anti-asialo GM1 (AGM1) antibody treatment did not increase the metastases in either organ. Interleukin-12 (IL-12) administration into mice induced strong cytotoxicities of NK cell-depleted liver and lung mononuclear cells (MNC) comparable to those without NK-cell depletion and inhibited metastases in either organ. In contrast, in both NK cell- and NK1+ int T-cell-depleted mice, IL-12 could not induce cytotoxic activity of liver and lung MNC and metastases in both organs increased with or without IL-12 treatment. These results confirmed the fact that NK+ int T cells are more potent antitumour effectors than NK cells against experimental haematogenous tumour metastases.