Logical modeling of thymus and natural killer lymphocyte differentiation

Thymus (T) and natural killer (NK) lymphocytes are important barriers against diseases. Therefore, it is necessary to understand regulatory mechanisms related to the cell fate decisions involved in the production of these cells. Although some individual information related to T and NK lymphocyte cell fate decisions have been revealed, the related network and its dynamical characteristics still have not been well understood. By integrating individual information and comparing with experimental data, we construct a comprehensive regulatory network and a logical model related to T and NK lymphocyte differentiation. We aim to explore possible mechanisms of how each lineage differentiation is realized by systematically screening individual perturbations. When determining the perturbation strategies, the state transition can be used to identify the roles of specific genes in cell type selection and reprogramming. In agreement with experimental observations, the dynamics of the model correctly restates the cell differentiation processes from common lymphoid progenitors to CD4+ T cells, CD8+ T cells, and NK cells. Our analysis reveals that some specific perturbations can give rise to directional cell differentiation or reprogramming. We test our in silico results by using known experimental observations. The integrated network and the logical model presented here might be a good candidate for providing qualitative mechanisms of cell fate specification involved in T and NK lymphocyte cell fate decisions.Supplementary informationThe online version contains supplementary material available at 10.1007/s10867-021-09563-y.     

Cellular tumorigenicity in nude mice. Role of susceptibility to natural killer cells

The athymic nude mouse is a useful animal model for assaying the neoplastic growth potential in vivo of animal cells transformed in vitro. Despite the demonstrated absence of thymus-dependent immunological functions, however, the nude mouse has now been shown to reject transplants of certain highly malignant heterologous tumors. In addition, a few transformed mammalian cell lines that exhibit all or most of the cellular phenotypes usually associated with malignancy fail to grow as tumors when injected into nude mice. In a continuing study to identify the in vitro phenotypes associated with tumor-forming ability in vivo, we investigated the role of cellular susceptibility to the naturally occurring, thymus-independent lymphocytes (natural killer or NK cells) in determining tumor induction by animal cells in nude mice. A representative collection of animal cells (ranging from normal human diploid cell strains to highly tumorigenic clonal cell lines, either transformed in vitro or derived from experimental tumors) was tested to see if the ability of cells to form tumors is consistently correlated with their susceptibility to NK cell-mediated lysis measured in vitro with splenic leukocytes from nude mice. If the physiological role of the NK cells in vivo were to recognize, and possibly to destroy, incipient tumor cells in situ, a direct association between cellular tumorigenicity and susceptibility to NK activity, might be expected. If, on the other hand, the formation of growing tumors by animal cells in nude mice depended on their ability to escape the cytolytic activity of NK cells, cellular tumorigenicity would be associated with cellular resistance to NK cells. Results obtained in this study failed to confirm either of these associations. Thus, cellular suscepbibility to NK cells, at least as determined by direct cytotoxicity assay in vitro, is not a useful predictive indicator of cellular tumorigenicity in nude mice.     

Genetic control of the natural killer cell activity in SJL and other strains of mice

Murine natural killer (NK) cell activity against lymphoma targets can be classified into three major functional phenotypes, i.e., low, inducible, and high, according to the levels of endogeneous activity and the extent of augmentation by interferon (IFN) or IFN inducers, as previously described. The prototype strains identifying these three phenotypes are SJL, A.SW, and B10.S, respectively, all bearing the H-2s haplotype. In the present study, the genetic basis of the low phenotype of SJL mice was examined further. F1 hybrid offspring of crosses between SJL and a strain with the high NK phenotype (B10.S, B10.D2, B10, C3H/HeN, or D1.LP) invariably expressed the high NK phenotype, indicating recessiveness of the low phenotype. Crosses between SJL and another low NK strain, such as A/J, A/HeN, or I/St, resulted in offspring of either the inducible or the high NK phenotype. Such genetic complementation between the low NK pairs indicates that the low phenotype of SJL and that of the other strains have different genetic bases. F1 hybrid mice between SJL and an inducible strain, A/WySn, were inducible, but those between SJL and the second inducible strain, A.SW, had the high NK phenotype. Thus, the congenic A/WySn and A.SW have distinct genotypes resulting in the same inducible phenotype. According to analyses of the segregating offspring from backcrosses of (SJL X B10.S)F1 mice to SJL, a single gene difference is responsible for the low endogenous level of NK activity in SJL as opposed to the high endogenous level in B10.S, and that the difference in three genes accounts for the poor responsiveness of NK cells to IFN in SJL mice. Studies of the two congenic lines of SJL, i.e., SJL-Igha and SJL-nu, indicated that the Igh locus is irrelevant for the low NK phenotype of SJL, but the nu locus clearly is relevant; SJL mice homozygous for the nu allele were phenotypically inducible in contrast to the nu/+ or +/+ mice which are low. The nu gene homozygosity rendered SJL mice responsive to IFN, not only in NK activity against lymphomas but also in ADCC activity against antibody-coated lymphoma cells.     

Renewal of natural killer cells in mice having elevated natural killer cell activity

The present study was designed to examined the dynamics of splenic natural killer (NK) cells under two conditions of enhanced NK cell activity: (1) CBA/J mice given polyinosinic-polycytidylic acid (poly-I:C), an NK-cell-enhancing agent, and 62) untreated athymic nude (nu/nu) mice. The 'total NK cell activity' of the spleen (percentage specific lysis corrected for changes in organ cellularity) increased 5-fold and 2.7-fold after poly-I:C treatment for 1 day and 4 days, respectively. An injection of hydroxyurea (HU), a cell-cycle-toxic drug, given together with either poly-I:C or saline to CBA/J mice resulted in both cases in a 25% reduction in total NK cell activity 1 day later. This suggests that the renewal rate of nondividing NK cells is similar in poly-I:C-treated and saline-injected mice, and that the NK-enhancing effect of poly-I:C is not due to a stimulation of proliferation among NK cell precursors. HU administered simultaneously with poly-I:C or saline for 4 days eliminated NK cell activity in both cases, indicating that spleen NK cell activity is mediated almost entirely by newly formed (less than or equal to 4 days) cells. In nude mice, NK cell activity was assayed at various intervals after an HU depletion period of 2 days. NK depletion was initially more rapid in nu/nu mice than in control (nu/+) mice, although equally profound, and the subsequent recovery of NK cell activity after cessation of HU was also more rapid than in control (nu/+) mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of the thymus in the maintenance of natural killer cells in vivo

This report describes a model for investigating the role of the thymus in regulating natural killer (NK) cell activity in vivo. Evidence is presented that the thymus can regulate NK cells, and that at least some NK cells can develop without thymic help. Marrow from thymectomized rats depleted of circulating T cells by thoracic duct cannulation was transplanted into rats without a thymus (1 degree ATX.BM). These 1 degree ATX.BM rats had NK cell levels above controls 3 months after reconstitution but markedly depressed NK cell levels by 9 months. When 1 degree ATX.BM marrow was used to reconstitute rats with or without a thymus, those without a thymus (2 degrees ATX.BM) exhibited low NK cell levels after 3 months, and a similar result was obtained when 2 degrees ATX.BM marrow was used to reconstitute 3 degrees ATX.BM rats. The low NK cell levels in 2 degrees and 3 degrees ATX.BM rats were due to a deficiency in spontaneously cytotoxic NK cells, as they had normal numbers of interferon-responsive pre-NK cells. Spleen cells from 2 degrees and 3 degrees ATX.BM rats produced less interferon than control spleen cells when cultured with P815 tumor cells in vitro. However, 2 degrees and 3 degrees ATX.BM rats had higher numbers of large granular lymphocytes than controls despite their low NK cell levels. In marked contrast to 2 degrees and 3 degrees ATX.BM rats, spleen cells from 4 degrees ATX.BM rats had higher levels of cytotoxicity and a higher frequency of both spontaneously cytotoxic and pre-NK cells than controls. The 4 degrees ATX.BM rats also had the highest frequency of large granular lymphocytes in the spleen.     

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.     

Suppression of B-cell differentiation by natural killer (asialo GM1+) cells in mice

Mice were injected intravenously with rabbit antiserum to ganglio-n-tetraosylceramide (asialo GM1, ASGM1), a neutral glycosphingolipid present at high quantities on the surface of natural killer (NK) cells. Spleen cells prepared from the mice were then examined for NK activity against YAC-1 targets, for phagocytic cells and by flow cytometric analysis for Thy1, Lyt1, Lyt2, ASGM1 and surface Ig (SIg) phenotypes. Administration of anti-ASGM1 in mice resulted in a complete depletion of NK activity and ASGM+1 cells in the spleen, but no changes in the proportions of Thy1+ cells and their Lyt1+ and Lyt2+ subsets and phagocytic cells. Corresponding to this selective depletion of ASGM+1 cells and NK activity, the spleen cells showed an increased number of SIg+ B cells and augmented mitogenic responses to B-cell but not T-cell mitogens. These NK-depleted spleen cells also showed production of pokeweek mitogen (PWM)-driven plaque-forming cells (PFC) to much higher levels than those of control spleens. In the spleens of mice treated with varying concentrations of anti-ASGM+1, a good correlation was found between the decreased NK activity and the enhanced PFC response. To directly test the possible suppressor activity of NK cells on PWM-induced PFC response, NK (ASGM+1) cells were highly purified from the spleen by a combination of Percoll gradients and cytolysis of T cells by monoclonal antibodies followed by indirect panning. When added to NK-depleted spleen cells, they suppressed the augmented PFC response of NK-depleted spleen cells, depending on the number of cells added. These results suggest that NK (ASGM+1) cells in mice exhibit a suppressor property on B cells, which are undergoing spontaneous or mitogen-induced differentiation.     

In vitro generation of natural killer cells from SJL/J bone marrow precursors

The development of natural killer (NK) cells from undifferentiated bone marrow (BM) precursors of low-NK-reactive SJL/J mice was studied. Results indicate that BM cells of untreated mice are not able to generate NK effector cells in cultures supplemented with recombinant interleukin-2 (IL-2). On the other hand in the presence of IL-2, NK cells are generated in cultures of BM from mice pretreated with 5-fluorouracil (5-FU, 150 mg/kg iv 4 days before harvesting), a treatment which has been shown to eliminate more differentiated but spare less differentiated BM precursors. The 5-FU resistant BM progenitor is asialoGM1-, Thy.1+, Lyt.1- and Lyt.2-. The cells generated by culturing with IL-2 are asialoGM1+, Thy.1+, Lyt.5+, Lyt.1-, Lyt.2- and lyse only NK-susceptible targets. Generation of NK cells is blocked by addition of anti-IL-2 receptor (IL-2/r) antibodies. These studies demonstrate that it is possible to generate NK effectors from SJL/J BM cells by in vitro culturing with IL-2.     

Defective interleukin-1 production by natural killer cells of patients with cancer

Summary Large granular lymphocytes (LGLs) from patients with malignant disease and from controls were activated by endotoxin or K562 cells, and the supernatants assayed for interleukin-1 (IL-1) activity. Normal LGLs produced significant amounts of IL-1, the activity of which could be neutralized by anti-human IL-1 antiserum. In patients with advanced cancer depressed IL-1 production was observed, which generally correlated with the degree of cytotoxicity produced by the LGLs. Prior treatment of the LGLs with interferon increased production of IL-1 by both control and patient cells. It is suggested that LGLs coming into contact with K562 cells produce IL-1, which is important in the effector-target cell interaction. The decreased cytotoxic activity of LGLs from cancer patients could be related to a defect in IL-1 production, an effect which can be partially corrected by in vitro interferon treatment.Abbreviations IL-1 Interleukin-1 - LGLs large granular lymphocytes - NK cells natural killer cells - IFN interferon - IL-2 interleukin-2 - HCC hepatocellular carcinoma - MN mononuclear cells - LPS lipopolysaccharide Supported in part by grants from the South African Medical Research Council, the National Cancer Association of South Africa, and the South African Chamber of Mines     

Tumor cell differentiation modulates susceptibility to natural killer cells

Induced differentiation in three human cell lines altered their sensitivity specifically to human natural killer (NK) cells by affecting their expression of NK target antigens. Differentiation of HL-60, a promyelocytic leukemia cell line, and the erythroleukemic cell line K562 was accompanied by a concomitant decrease in susceptibility to NK-mediated lysis whereas induction of MeWo melanoma cells resulted in an enhanced sensitivity to lysis. Our findings suggest that target cell susceptibility to NK-mediated lysis may in part be dependent on the stage of differentiation of the tumor cell target.     

Regulation of B lymphocytes by natural killer cells. Role of IFN-gamma

Using a co-culture system of fractionated B cells and highly purified NK cells, we have demonstrated direct interactions between B lymphocytes and NK cells. B cells are able to stimulate the production of IFN-gamma by NK cells. This stimulatory ability is restricted to a subpopulation of large, presumably in vivo activated B lymphocytes. The secreted IFN-gamma in turn inhibits polyclonally induced B cell proliferation. Small resting B cells neither stimulate IFN-gamma production nor are they measurably affected by NK cells.     

Carbohydrates in the functions of natural killer cells.

There is little doubt that carbohydrates are crucial to the functions of NK cells. Exogenous carbohydrates alter the cytolytic capabilities of these cells, treatment of NK cells with glycosidases or inhibitors of glycosylation affect function, NK cells can bind selective sugars, and NK cells can be identified and subdivided into functionally distinct subsets on the basis of cell surface carbohydrates. Yet, despite the large number of observations which have been made concerning carbohydrates and NK cells, there is little consensus regarding these studies and few investigations which satisfactorily demonstrate specific mechanisms for the observed effect of the carbohydrates. Almost certainly, the number of diverse observations implies roles for carbohydrates in multiple areas of NK function, probably including target recognition, tissue distribution and post-binding events in the lytic cascade. Hopefully, these observations made to date will be viewed as exciting preliminary studies which will entice more sophisticated investigations designed to elucidate the precise roles of carbohydrates in the functions of NK cells.     

Role of histamine in natural killer cell-mediated resistance against tumor cells

The formation of lung metastases by i.v.-injected B16 melanoma (F1 and F10 strain) cells in Swiss albino, C57BL/6, and BALB/c mice was reduced by a single dose of histamine given 24 h before tumor cell inoculation. The antimetastatic effect of histamine was specifically mediated by histamine H2-receptors (H2R): it was blocked by the H2R antagonist ranitidine and mimicked by dimaprit, a specific H2R agonist but not by an H2R-inactive structural analog of this compound, nor-dimaprit, or the H1R agonist 2-thiazolyl-ethylamide. A single dose of any of the H2R antagonists ranitidine, tiotidine, famotidine, or cimetidine drastically augmented metastasis. Effects of H2R-interactive compounds on B16 metastasis required intact NK cells, as judged by the inability of histamine or ranitidine to affect B16 metastasis after NK cell depletion in vivo using antibodies to asialo-GM1. NK-cell-mediated lysis of YAC-1 lymphoma cells in vivo was enhanced by histamine and reduced by ranitidine within 4 h after inoculation of tumor cells. The antimetastatic effect of IL-2 was potentiated by histamine; in some experiments, combined treatment with a low dose of IL-2 (6000 U/kg) and histamine completely eliminated metastasis, whereas concomitant treatment with ranitidine abrogated antimetastatic effects of IL-2; animals treated with ranitidine and IL-2 displayed the same level of enhanced metastasis as those treated with ranitidine alone. The presented data are suggestive of an earlier unrecognized role for histamine in NK cell-mediated resistance against metastatic tumor cells.     

Suppressive effects of monocytic cells and transforming growth factor-beta on natural killer cell differentiation in autoimmune viable motheaten mutant mice.

Viable motheaten (mev) mice are homozygous for a recessive single gene mutation at chromosome 6. These mice develop numerous inflammatory and arthritic syndromes and exhibit abnormal B cell functions as well as lower T and NK cell activity. In this study, the differentiation of NK cells in mev mice was examined to elucidate the underlying basis for decreased NK activity. Although NK cells appear to be present in mev mice, their activity was demonstrable only when the spleen cells were enriched by nylon wool passage. Similarly bone marrow cells from these mice could be shown to contain precursors of NK cells when they were passed over nylon wool and transplanted into irradiated recipients. The adherent cells from both the spleen and bone marrow of mev mice suppressed the differentiation of NK cells from normal splenic populations. These suppressive adherent cells were F4/80(+), AsGm-1(+), Qa-5(+), and NK-1.1(+). They were not cytolytic when cultured in IL-2. Antibodies to a number of cytokines, such as IFN-alpha, -beta, and gamma, or TNF-alpha, could not reverse the suppressive effect of the adherent cells. Addition of anti-TGF-beta antibody could, however, overcome the suppression, suggesting that TGF-beta was partly responsible for the defective NK differentiation in the mev mice.     

Role of natural killer cells in resistance against friend retrovirus-induced leukemia

We have previously shown that immunization with a synthetic peptide that contains a single CD4(+) T-cell epitope protects mice against immunosuppressive Friend retrovirus infection. Cells producing infectious Friend virus were rapidly eliminated from the spleens of mice that had been immunized with the single-epitope peptide. However, actual effector mechanisms induced through T-helper-cell responses after Friend virus inoculation were unknown. When cytotoxic effector cells detected in the early phase of Friend retrovirus infection were separated based on their expression of cell surface markers, those lacking CD4 and CD8 but expressing natural killer cell markers were found to constitute the majority of effector cells that lysed Friend virus-induced leukemia cells. Depletion of natural killer cells by injecting anti-asialo-ganglio-N-tetraosylceramide antibody did not affect the number of CD4(+) or CD8(+) T cells in the spleen, virus antigen-specific proliferative responses of CD4(+) T cells, or cytotoxic activity against Friend virus-induced leukemia cells exerted by CD8(+) effector cells. However, the same treatment markedly reduced the killing activity of CD4(-) CD8(-) effector cells and completely abolished the effect of peptide immunization. Although the above enhancement of natural killer cell activity in the early stage of Friend virus infection was also observed in mice given no peptide, these results have demonstrated the importance and requirement of natural killer cells in vaccine-induced resistance against the retroviral infection.     

Adenovirus persistence in man. Defective E1A gene product targeting of infected cells for elimination by natural killer cells

Human adenovirus types 2 and 5 (Ad2/5) cause persistent infections in man. Ad2/5 infection of rodent cells induces increased susceptibility to NK lymphocyte-mediated lysis that is dependent on target cell expression of Ad2/5 E1A gene products. In contrast to infected rodent cells, Ad2/5 infection of human fibroblasts and epithelial cells does not result in increased susceptibility to either human or rodent NK cell-mediated killing, despite high levels of E1A protein expression. This functional inactivity of E1A gene products in Ad-infected human cells may contribute to adenoviral persistence by rendering the NK cell response to Ad-infected cells ineffective.     

Participation of natural killer cells in the recovery of mice from visceral leishmaniasis

After infection with the protozoan parasite Leishmania donovani, C57BL/6J bg/bg (beige) mice, which are deficient in natural killer (NK) activity, were unable to control splenic parasite loads relative to phenotypically normal C57BL/6J bg/+ and +/+ mice, particularly beyond 21 days of infection. When beige mice were injected intravenously with 2 or 3 X 10(6) syngeneic, cloned NK cells (NKB61B10 cell line), they displayed splenic parasite burdens which did not differ significantly from those of normal controls. In C57BL/6 +/+ mice rendered NK deficient by split-dose irradiation (four weekly, 200-rad doses of gamma irradiation beginning at 4 weeks of age) splenic and hepatic parasite levels were significantly higher than those in nonirradiated controls at 15 days of infection and beyond. In both sets of experiments, relative degrees of hepato- and splenomegaly were not sufficient to account for differences in parasite burdens among NK-deficient and normal mice. Taken together, the results of these experiments suggest that NK cells may contribute to parasite elimination during the acquired-resistance phase of L. donovani infection in mice.     

Role of natural killer T cells in host defence against cryptococcal infection

Cryptococcosis is an opportunistic fungal infectious disease that often occurs in severely immunocompromised patients. Host defence against the causative microorganism is largely mediated by cellular immunity, and Th1 cytokines, such as IFN-gamma, play central roles in the host protective responses. IL-12 and IL-18 activate the synthesis of IFN-gamma by innate immune cells, including NK, NKT and gamma delta T cells and promote the differentiation of Th1-type acquired immune responses. Recently, NKT cells, which are involved in the recognition of glycolipid antigens, have attracted much attention based on their potent immunomodulating activities. Several studies have reported the role of this particular component of innate immune responses in tumor immunity and pathogenesis of autoimmune diseases. In this review, I outline the recent findings on the role of NKT cells in host defence against infectious microorganisms, with a special focus on our data emphasizing the importance of this subset of immunocytes in the development of acquired as well as early host protection against cryptococcal infection.