Thymic Natural Killer Research Articles

Thymic NK-Cells and Their Potential in Cancer Immunotherapy.

Natural killer (NK)-cells are innate immune cells with potent anti-tumor capacity, capable of recognizing target cells without prior exposure. For this reason, NK-cells are recognized as a useful source of cell therapy. Although most NK-cells are derived from the bone marrow (BM), a separate developmental pathway in the thymus also exists, producing so-called thymic NK-cells. Unlike conventional NK-cells, thymic NK (tNK)-cells have a combined capacity for cytokine production and a natural ability to kill tumor cells in the presence of NK-cell receptor stimulatory ligands. Furthermore, tNK-cells are reported to express CD3 subunits intracellularly, without the presence of a rearranged T-cell receptor (TCR). This unique feature may enable harnessing of these cells with a TCR to combine NK- and T-cell effector properties in one cell type. The development, phenotype, and function of tNK-cells, and potential as a cell therapy is, however, poorly explored. In this review, we provide an overview of current literature on both murine and human tNK-cells in comparison to conventional BM-derived NK-cells, and discuss the potential applications of this cellular subset in the context of cancer immunotherapy.

TRAF3IP3 at the trans-Golgi network regulates NKT2 maturation via the MEK/ERK signaling pathway.

Thymic natural killer T (NKT)2 cells are a subset of invariant NKT cells with PLZFhiGATA3hiIL-4+. The differentiation of NKT2 cells is not fully understood. In the present study, we report an important role of TRAF3-interacting protein 3 (TRAF3IP3) in the functional maturation and expansion of committed NKT2s in thymic medulla. Mice with T-cell-specific deletion of TRAF3IP3 had decreased thymic NKT2 cells, decreased IL-4-producing peripheral iNKTs, and defects in response to α-galactosylceramide. Positive selection and high PLZF expression in CD24+CD44- and CCR7+CD44- immature iNKTs were not affected. Only CD44hiNK1.1- iNKTs in Traf3ip3-/- mice showed reduced expression of Egr2, PLZF, and IL-17RB, decreased proliferation, and reduced IL-4 production upon stimulation. This Egr2 and IL-4 expression was augmented by MEK1/ERK activation in iNKTs, and TRAF3IP3 at the trans-Golgi network recruited MEK1 and facilitated ERK phosphorylation and nuclear translocation. LTβR-regulated bone marrow-derived nonlymphoid cells in the medullary thymic microenvironment were required for MEK/ERK activation and NKT2 maturation. These data demonstrate an important functional maturation process in NKT2 differentiation that is regulated by MEK/ERK signaling at the trans-Golgi network.

Nfil3 Is a Glucocorticoid-Regulated Gene Required for Glucocorticoid-Induced Apoptosis in Male Murine T Cells

Glucocorticoids (GCs) have essential roles in the regulation of development, integrated metabolism, and immune and neurological responses, and act primarily via the glucocorticoid receptor (GR). In most cells, GC treatment results in down-regulation of GR mRNA and protein levels via negative feedback mechanisms. However, in GC-treated thymocytes, GR protein levels are maintained at a high level, increasing sensitivity of thymocytes to GCs, resulting in apoptosis termed glucocorticoid-induced cell death (GICD). CD4(+)CD8(+) double-positive thymocytes and thymic natural killer T cells in particular are highly sensitive to GICD. Although GICD is exploited via the use of synthetic GC analogues in the treatment of hematopoietic malignancies, the intracellular molecular pathway of GICD is not well understood. To explore GICD in thymocytes, the authors performed whole genome expression microarray analysis in mouse GR exon 2 null vs wild-type thymus RNA 3 hours after dexamethasone treatment. Identified and validated direct GR targets included P21 and Bim, in addition to an important transcriptional regulator Nfil3, which previously has been associated with GICD and is essential for natural killer cell development in vivo. Immunostaining of NFIL3 in whole thymus localized NFIL3 primarily to the medullary region, and double labeling colocalized NFIL3 to apoptotic cells. In silico analysis revealed a putative GC response element 5 kb upstream of the Nfil3 promoter that is strongly conserved in the rat genome and was confirmed to bind GR by chromatin immunoprecipitation. The knockdown of Nfil3 mRNA levels to 20% of normal using specific small interfering RNAs abrogated GICD, indicating that NFIL3 is required for normal GICD in CTLL-2 T cells.

GATA-3 is required for early T lineage progenitor development

Most T lymphocytes appear to arise from very rare early T lineage progenitors (ETPs) in the thymus, but the transcriptional programs that specify ETP generation are not completely known. The transcription factor GATA-3 is required for the development of T lymphocytes at multiple late differentiation steps as well as for the development of thymic natural killer cells. However, a role for GATA-3 before the double-negative (DN) 3 stage of T cell development has to date been obscured both by the developmental heterogeneity of DN1 thymocytes and the paucity of ETPs. We provide multiple lines of in vivo evidence through the analysis of T cell development in Gata3 hypomorphic mutant embryos, in irradiated mice reconstituted with Gata3 mutant hematopoietic cells, and in mice conditionally ablated for the Gata3 gene to show that GATA-3 is required for ETP generation. We further show that Gata3 loss does not affect hematopoietic stem cells or multipotent hematopoietic progenitors. Finally, we demonstrate that Gata3 mutant lymphoid progenitors exhibit neither increased apoptosis nor diminished cell-cycle progression. Thus, GATA-3 is required for the cell-autonomous development of the earliest characterized thymic T cell progenitors.

Identification of a Common Developmental Pathway for Thymic Natural Killer Cells and Dendritic Cells

Current data support the notion that the thymus is seeded by a yet uncommitted progenitor cell able to generate T cells, B cells, natural killer (NK) cells, and dendritic cells (DCs). We assess in this report the developmental relationship of DCs and NK cells derived from a small subset of CD34(+) human postnatal thymocytes that, like the earliest precursors in the fetal thymus, display low CD33 surface expression. Culture of these isolated CD34(+) CD33(lo) thymic progenitors with a mixture of cytokines, including interleukin-7 (IL-7), IL-1alpha, IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, results in predominant generation of DCs. However, the addition of IL-2 to the cytokine mixture leads to the simultaneous development of DCs and NK cells. Both developmental pathways progress through a transient population of CD34(+)CD44(bright) CD5(lo/-)CD33(+) large-sized cells, distinct from small-sized T-lineage precursors, that contain bipotential NK/DC progenitors. These data provide evidence of linked pathways of NK cell and DC development from intrathymic precursors and suggest that NK cells and DCs branch off the T lineage through a common intermediate progenitor.

Association between the degree of thymic dysplasia and the kinetics of thymic NK cell activity during the graft-versus-host reaction

In this study, by employing different cell doses and parent into F 1 hybrid combinations, we have investigated the relationship between the severity of thymic medullary dysplasia and the kinetics of thymic natural killer (NK) cell activity after the induction of graft-versus-host (GVH) reactions. GVH reactions were induced by injecting different doses (30, 20, 10 × 10 6) of C57BL 6 (B6) of A parental lymphoid cells (PLC) into non-X-irradiated adult B6xAF 1 (B6AF 1) mice. On different days after the induction of GVH reactions, the thymuses were examined histologically and thymocyte NK cell activity was tested by using YAC targets. Our results show that, depending upon the genotype and dose of PLC injected, various degrees of thymic medullary dysplasia (mild, moderate, or severe) can be induced. Furthermore, severe to moderate thymic medullary dysplasia is observed only in those groups of GVH-reactive mice in which thymic NK cell activity occurs early and increases rapidly. In contrast, when mild thymic medullary dysplasia or no thymic alterations was observed, thymic NK cell activity peaked later and was of lower intensity than that of the groups with moderate to severe lesions. These results suggest an association between the degree of thymic medullary dysplasia and the kinetics of NK cell activity in the thymus. Furthermore, the different degrees of thymic medullary dysplasia as described here may serve as a powerful tool to study the role of thymic medullary dysplasia in determining the duration of T-cell immunodeficiency associated with the GVH reactions.

Natural killer cells in murine muscular dystrophy: IV. Characterization of percoll fractionated splenic and thymic natural killer cells and natural killer-sensitive thymocyte targets

The Natural Killer (NK) activity in the thymus and NK-sensitive thymocyte targets of dystrophic mice was investigated. Dystrophic and normal mouse thymocytes or spleen cells were layered on discontinuous Percoll gradients (5 or 10% increments, respectively) between 40 and 70% and centrifuged at 1700g for 30 min. All fractions were tested for either NK activity or used as 51Cr-labeled NK-sensitive targets in a 6-hr 51Cr release assay. The density interface between the 50% (1.060 g/ml) and 60% (1.075 g/ml) Percoll fractions of either dystrophic or normal mouse spleen cells and the 40% (1.050 g/ml) and 50% (1.060 g/ml) Percoll fractions of either dystrophic or normal mouse thymocytes were found to contain the largest proportion of NK activity using YAC-1 lymphoma tumor cells as targets. In addition, the NK activity in dystrophic mouse spleen cells and thymocytes was significantly greater when compared with normal mouse controls. Target binding cell studies revealed that these Percoll fractions of dystrophic mouse spleen cells and thymocytes had greater numbers of conjugate-forming cells when compared with normal control groups. Cell depletion experiments using either anti-Thy 1.2, anti-asialo-GM 1 or anti-NK-1 plus complement treatment revealed that the cell responsible for NK activity in the 50% Percoll fraction interface of dystrophic mouse spleen cells was asialo-GM 1 positive, NK-1 positive, and partially Thy 1.2 positive. However, the cells displaying NK-activity in the thymus of normal or dystrophic mice were found to be highly Thy-1.2 positive and peanut agglutinin (PNA) negative. The density interface between the 60% (1.075 g/ml) and 65% (1.081 g/ml) Percoll fractions of either normal or dystrophic mouse thymocytes contained the largest proportion of NK-sensitive target cells. Interestingly, the 60% Percoll fraction of dystrophic mouse thymocyte targets was significantly more susceptible to NK-mediated lysis than that of the normal mouse thymocyte population. Cell depletion experiments revealed that the NK-sensitive thymocyte population was similar in both mice, that is. Thy-1.2 positive, cortisone sensitive, PNA positive. Dolichos biflorus (DBA) negative and asialo GM-1 negative. The results indicate that there are density differences between splenic and thymic NK cells. In addition, there are density and phenotypic differences between thymic NK cells and thymic NK-sensitive target cells. The findings support the hypothesis that there are different populations of NK cells.

Studies of the thymus in mice bearing the Lewis lung carcinoma: I. Thymic natural killer cell activity in 3LL tumor-bearing mice

Natural killer (NK) cell activity of spleen and thymus was studied in Lewis lung carcinoma (3LL)-bearing mice. A high level of hydrocortisone-sensitive NK cell cytotoxicity was found in the thymus after excision of the primary tumor, but not in nonamputated mice. Conversely, splenic NK cell activity was diminished in nonamputated tumor-bearing mice and remained unmodified in amputated tumor-bearing mice. Excision of the primary tumor prevented tumor-induced thymic atrophy and splenomegaly. Histological study of the thymus revealed the presence of metastatic cells within the cortex. These data suggest that NK cells or their precursors are present in the thymus and can be triggered or unmasked in situ by tumor cells migrating from the primary tumor.

Studies of the thymus in mice bearing the Lewis lung carcinoma: II. Modulation of thymic natural killer activity by thymulin (FTS-Zn) and the antimetastatic effect of zinc

Modulation of thymic natural killer (NK) cell activity by the serum thymic factor (thymulin or FTS-Zn) was demonstrated in mice carrying the Lewis lung carcinoma (3LL). Thymulin decreased the increased NK activity found in amputated (tumor) tumor-bearing mice and induced a significant NK activity in nonamputated mice, normally devoid of such activity. Zinc had no effect in either case. Histological studies revealed that zinc prevented the tumor-induced thymic atrophy. A clear-cut decrease in the number of lung metastases was observed in zinc-treated mice. This effect was suppressed by concomitant administration of thymulin to amputated mice. The thymic dependency of the antimetastatic action of zinc as well as the mode of action of thymulin on NK cell activity in tumor-bearing animals is discussed.