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.

Pan natural killer cell monoclonal antibodies and their relationship to the NK1.1 antigen.

The study of natural killer (NK) has been difficult because they account for a small percentage of peripheral blood and splenic lymphocytes and the paucity of NK specific antigens that have been identified. We have isolated pure populations of C57BL/6 (H-2b) NK cells using the IgG2b monoclonal antibody PK136 (anti-NK1.1). These NK1.1+ cells were used to immunize 129/J (H-2b) mice, and in this report, we describe three new NK specific monoclonal antibodies (SW3A4(IgM), SW4B12(IgG1), and SW2B4(IgG2b] and their relationship to the known murine NK antigen NK1.1. We have further characterized the NK1.1 antigen as a 39 kd molecule which is coded for by a gene which appears to map to chromosome 6.

Murine natural killer cells express functional Fc gamma receptor II encoded by the Fc gamma R alpha gene.

We report evidence that murine NK cells express a functional Fc gamma RII encoded by the Fc gamma RII alpha gene. Several lines of indirect evidence indicate that freshly obtained NK cells from mice of several strains bear a functional Fc gamma RII: (a) anti-Fc gamma RII antibody 2.4G2 detects a small but significant proportion of sIg- cells and a small proportion of the 2.4G2+ cells are included in the Thy-1+ population; (b) sIg- lymphocytes contain 2.4G2+ and Fc gamma R-bearing cells in similar proportions; (c) binding of particulate immune complexes by sIg- lymphocytes is completely inhibited by 2.4G2; (d) 2.4G2+ cells mediate greater than 50% of the spontaneous cytotoxicity in sIg- splenic lymphocytes. Direct evidence for the presence of Fc gamma RII on murine NK cells is provided by the results of two-color immunofluorescence studies performed on splenic lymphocytes from C57BL/6 mice showing coexpression of NK-1.1 and 2.4G2. Studies of in vitro propagated homogeneous NK cell populations confirm that murine NK cells express only Fc gamma RII and that this Fc gamma R is functional, as shown in experiments of inhibition of ADCC by the anti-Fc gamma RII antibody 2.4G2. The results of studies at the molecular level show that an Fc gamma RII alpha transcript identical to that expressed in macrophages is the only molecule encoding Fc gamma RII in murine NK cells.

Natural killer cells and their precursors in mice with severe combined immunodeficiency.

Our studies with scid mice have clarified the relationship between T cells and NK cells. C.B-17 scid mice have normal frequency of transplantable NK progenitors in their bone marrow which develop into fully functional NK cells. Spleens of scid mice contain mature NK cells which are phenotypically and functionally indistinguishable from NK cells found in normal mice. These cells retain their TCR genes in germline configuration and do not transcribe the CD3 genes. Thus, NK cells are distinct from the earliest identifiable cells committed to the T-lineage. In addition to the spleen, the thymus of scid mice also contains mature NK cells. These cells constitute a small proportion of the thymus cell population and can be clearly distinguished from the majority of cells, which have the phenotype and molecular characteristics of very early T-lineage cells. There is no evidence that NK cells within the thymus are derived in situ from a common NK/T precursor. Together these data support the hypothesis that NK cells form an independent lineage.

Murine natural killer cells stimulated in vivo do not express the T cell receptor alpha, beta, gamma, T3 delta, or T3 epsilon genes.

Murine blast natural killer (NK) cells responding to in vivo stimulation were isolated and characterized as to their expression of the T cell receptor (TCR) variable genes alpha, beta, and gamma and the TCR constant genes T3 delta and T3 epsilon. In vivo stimulated blast T cells were isolated for comparison. RNA extracted from highly purified blast cell populations elicited in vivo was probed for TCR transcripts by Northern blot analysis. In contrast to the blast T cells which expressed high levels of the alpha, beta, delta, and epsilon genes, blast NK cells expressed very low to not detectable levels of these genes. Blast NK cells isolated from euthymic mice were comparable to those isolated from athymic mice and both populations had profoundly reduced levels of transcripts for TCR genes. The expression of gamma-chain genes was extremely low in the in vivo elicited blast T cells as well as the blast NK cells. Highly purified NK cells isolated from unmanipulated mice and propagated in culture with recombinant interleukin 2 for short periods of time also had extremely low levels of delta and epsilon, whereas T cells expanded in culture had high level expression of these genes. As delta and epsilon appear to be required for expression of a functional recognition structure on the T cell surface and are expressed early in T cell ontogeny, these results indicate that the functional NK cells responding to proliferation signals do not form a conventional T cell recognition structure. Furthermore, the results support a separation of the T cell and NK cell lineages early in ontogeny.

T cell receptor genes do not rearrange or express functional transcripts in natural killer cells of scid mice.

The lineage of natural killer (NK) cells is poorly understood. To examine the relationship between NK cells and cells of the T lineage we have examined purified NK cells from a mutant mouse strain (scid) with severe combined immunodeficiency. Approximately 40% of the lymphoid cells in the spleens of scid mice expressed the NK-specific marker NK-2.1. All NK activity of the scid spleen cells could be accounted for by the NK-2.1+ population, similar to results obtained by using normal C57BL/6 X DBA/2 (B6D2F1) mice. Sorted NK-2.1+ cells proliferated in response to human recombinant interleukin 2 (r-IL 2) but not to concanavalin A (Con A), and were maintained in culture for 2 to 3 wk. Cultured NK-2.1+ cells displayed a cell surface phenotype (Asialo-GM1+, Thy-1.2+, L3T4-, Lyt-2-) and lytic activity similar to that described for freshly isolated NK cells of normal mice. Furthermore, T cell receptor (TCR) genes of the TCR-gamma and TCR-beta loci were in germline configuration, and no functional transcripts of TCR-gamma, TCR-beta, or TCR-alpha were detected. We propose that the expression of the TCR is not necessary for functional NK activity, and NK cells are distinct from both mature cytotoxic T lymphocytes and the earliest identifiable T cells.