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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 NKl.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 CD5 dim and 20–25% of which expressed TCR-β8 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 tjiat 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.

The Ly24 (Pgp-1) marker is expressed on some, but not all, mature T lymphocytes. It has recently become apparent that the development of Ly24− T lymphocytes is dependent on the presence of an intact thymus and that virgin Ly24− T cells rapidly acquire this marker upon antigenic or mitogenic stimulation. Although natural killer (NK) cells can develop and function in the absence of an intact thymus, some NK cell subsets express certain markers normally associated with T lymphocytes. The experiments in this report were undertaken to determine if NK cells express Ly24 and whether such an expression could be used to delineate distinct NK cell subsets. We found that mature functional NK cells expressed the Ly24 marker as defined by the monoclonal antibody 9F3. Double-color fluorescence analysis using C57BL/6 splenocytes (whose NK cells express the NK1.1 marker) showed all the NK1.1+ cells to be Ly24+ as well. For C3H/HeN (an NK 1.1− strain), double-color fluorescence analysis utilizing asialo GM1 and Ly24 revealed a distinct subset positive for both markers and containing most of the functional NK cell activity. Whereas the Ly24 marker did not illuminate an NK cell subset, these findings demonstrate that this determinant can be useful for the further characterization and isolation of NK cells.

In an attempt to characterize the “activation” signal delivered by concanavalin A (Con A) in lectin-dependent T-cell-mediated cytotoxicity (LDCC), we attempted to determine whether there was a need for the intracellular localization of lectin within the effector cell. To preclude such internalization, Con A was insolubilized by coupling to Sepharose and was then tested for its ability to support LDCC. We found that all the preparations of Con A-Sepharose tested were effective in supporting cytolysis, suggesting that the “activation” signal for lysis could be delivered solely at the level of the effector cell membrane. Furthermore, in contrast to previous reports, we found that insolubilized Con A was mitogenic for T-cell populations.

Prior work has suggested that Mabs against the transferrin receptor (ATRAs) may function as selective inhibitors of lymphocyte activation and that T cell activation protocols may be more sensitive to ATRA-mediated inhibition than B cell activation protocols. New side-by-side functional comparisons of three ATRAs are presented. When these studies are considered with our prior work they demonstrate unambiguously that although one particular IgG ATRA consistently fails to inhibit LPS responses and although IgM ATRAs may be slightly more effective inhibitors than IgG ATRAs, ATRAs as a class consistently appear to abolish the MLR at submicrogram concentrations, essentially eliminate cytotoxic cell generation at concentrations between 1 and 5 micrograms/ml, and produce no more than about 50% inhibition of LPS responses at concentrations as high as 25 micrograms/ml. Therefore, an even stronger case can now be made for the idea that lymphocyte subsets differ in their dependence on transferrin receptor function during activation. This, in turn, makes an even stronger case for the idea that lymphocyte subsets differ in fundamental aspects of the management of their iron economies. New studies also show that IgG ATRAs appear to function by causing down-modulation of surface expression of the transferrin receptor in normal lymphocytes in a manner similar to that previously shown for tumor cells. It is clear that a sophisticated model will ultimately be required to account for all of the data arising from studies with ATRAs, and a new attempt at a more detailed construct is presented.

We have previously reported that mouse bone marrow (BM) cells stimulated with alloantigen produce cytotoxic effector T-cell activity and produce interferon ( IFN -) α β . In this report we show evidence suggesting that interleukin 2 (IL-2) may play a role in this IFN- α β production by alloantigen-stimulated BM cells. Alloantigen-induced IFN production by bone marrow cells was completely inhibited when cultures were supplemented with antisera to IL-2. Cell-free supernatants obtained at 2 days from cultures containing CS7BL 6 BM cells and irradiated DBA 2J spleen cells were also shown to contain low levels of IL-2 activity and induced significant IFN production in fresh BM cells. Different IL-2 preparations were tested for their ability to induce IFN- α β production in mouse BM cells. Mouse BM cells cultured with recombinant human IL-2 or highly purified mouse IL-2 produced high levels of IFN- α β activity after 2–3 days of culture with significant IFN activity being detected as early as 24 hr of culture. IL-2-induced IFN- α β production was partially resistant to irradiation. In contrast, irradiated (2000 rad) bone marrow cells failed to produce any IFN when cultured with alloantigen in the absence of IL-2. T-cell-depleted BM cells or BM cells obtained from C57BL 10 nude mice produced high levels of IFN- α β following stimulation with IL-2. In addition, bone marrow cells depleted of Ia+, Qa 5+, or Asialo GM1+ cells produced IFN in response to IL-2. Thus, neither T cells nor NK cells are required for IL-2-induced IFN- α β production by BM cells. The action of IL-2 on bone marrow cells to induce IFN production was mediated by the classical IL-2 receptor, since monoclonal antibodies to the IL-2 receptor present on T cells blocked this response and since bone marrow cells depleted of IL-2 receptor-bearing cells failed to produce IFN when cultured with IL-2. These results suggest that non-T cells resident in the BM have receptors for IL-2 and can produce IFN- α β upon stimulation by IL-2. Since IFN has been shown to affect different aspects of hematopoiesis, the production of IFN by BM cells stimulated by IL-2 may be important in the control of hematopoiesis. In addition, IL-2-induced IFN production may play a role in graft-versus-host disease.

Spleen cells obtained from normal mice were cultured with interleukin 2 (IL2); no antigen was added. After 4–5 days, these cultures contained effector cells which lysed autologous spleen target cells that were modified with 2,4,6-trinitrobenzene sulfonic acid or fluorescein isothiocyanate. No killing was seen on unmodified spleen target cells. These effector cells were Thy 1+, Lyt 1−, 2+ and were derived from Thy 1+ precursor cells. IL2 preparations induced the generation of such cytotoxic T lymphocytes (CTL) in a dose-dependent manner. IL2-induced CTL were shown to be different from the natural killer (NK) cells augmented by IL2 by virtue of their time of appearance in culture, by cold-target competition, and by different cell-surface markers. These results demonstrate that the IL2 signal may be sufficient for the induction of the differentiation of CTL precursors in the absence of an antigenic signal.