Splenic NK1.1+CD4+ T cells that express intermediate levels of TCRαβ molecules (TCRint) and the DX5 Ag (believed to identify an equivalent population in NK1.1 allelic negative mice) possess the ability to rapidly produce high quantities of immunomodulatory cytokines, notably IL-4 and IFN-γ, upon primary TCR activation in vivo. Indeed, only T cells expressing the NK1.1 Ag appear to be capable of this function. In this study, we demonstrate that splenic NK1.1-negative TCRintCD4+ T cells, identified on the basis of FcγR expression, exist in naive NK1.1 allelic positive (C57BL/6) and negative (C3H/HeN) mice with the capacity to produce large amounts of IL-4 and IFN-γ after only 8 h of primary CD3 stimulation in vitro. Furthermore, a comparison of the amounts of early cytokines produced by FcγR+CD4+TCRint T cells with NK1.1+CD4+ or DX5+CD4+TCRint T cells, simultaneously isolated from C57BL/6 or C3H/HeN mice, revealed strain and population differences. Thus, FcγR defines another subpopulation of splenic CD4+TCRint cells that can rapidly produce large concentrations of immunomodulatory cytokines, suggesting that CD4+TCRint T cells themselves may represent a unique family of immunoregulatory CD4+ T cells whose members include FcγR+CD4+ and NK1.1/DX5+CD4+ T cells.

Aunique subpopulation of CD4+ T cells that coexpress the NK1.1 Ag, a member of NKR-P1 NK cell receptor family found on NK cells (1), and TCR has been identified at relatively high frequencies in the thymus (2), liver (3), and bone marrow (4). They are also present at lower frequencies in peripheral lymphoid organs such as the spleen and lymph node, where they account for 1–2% of the total T cell population (5). Unlike conventional T cells, NK1.1+CD4+ T cells express intermediate levels of TCR-CD3 molecules on their surface and have a very restricted TCR usage that is heavily biased for both α- and β-chains. Most NK1.1+CD4+ T cells use one of three Vβ chains (Vβ8, Vβ7, or Vβ2) paired with a single invariant Vα14 chain (Vα14-Jα281) (2, 6, 7). Despite existing in naive and pathogen-free mice, these cells express markers on their surface at levels usually associated with an activation or memory phenotype such as CD44high, 3G11low, and CD62Llow (5, 8).

Although the study of NK1.1+CD4+ T cells has been limited to mouse strains that carry the NK1.1 allele, such as C57BL/6 mice, it is assumed that equivalent cells exist in NK1.1-negative mouse strains. Recently, a newly identified Ag (DX5), expressed by the majority of NK1.1+CD4+ T cells (9), has been identified on small T cell subpopulations in NK1.1-negative mouse strains. It is believed that these DX5-positive T cell subsets are equivalent to NK1.1+CD4+ T cells.

NK1.1+CD4+ T cells can develop in the thymus and also in several extrathymic tissues such as the liver (10, 11). Unlike conventional CD4+ T cells whose thymic development is restricted by self MHC class II molecules, NK1.1+CD4+ T cells are selected and restricted by CD1, a β2-microglobulin-associated nonclassical MHC class I molecule (10, 12, 13, 14). Hence, these cells develop normally in MHC class II-deficient mice, but are markedly diminished in number in the thymus and spleen of β2-microglobulin (10) and CD1-deficient (13, 14) mice.

A unique characteristic of NK1.1+CD4+ T cells is their ability to produce large amounts of cytokines, in particular IL-4 and IFN-γ, upon primary TCR engagement in vitro (15, 16) and in vivo (17). Although NK1.1+CD4+ T cells stimulated in vitro secrete IL-4 and IFN-γ with conventional kinetics, being detectable at about 24 h (1), their stimulation in vivo by anti-CD3 mAb results in rapid IL-4 and IFN-γ mRNA induction and protein secretion, peaking at 90 min (17). Because of their capacity to rapidly produce large quantities of cytokines, particularly IL-4, it is thought that at the onset of an immune response these cells are responsible for directing the development of naive CD4+ T cells into Th2 cells (17). The findings that both β2-microglobulin-deficient and SJL mice, which have diminished numbers of NK1.1+CD4+ T cells, have a reduced capacity to produce IgE in response to in vivo stimulation with anti-IgD (18, 19) support this hypothesis. Also, mice transgenic for TCR Vα14 chain have an increased frequency of splenic NK1.1+CD4+ T cells and elevated basal levels of serum IgG1 and IgE (20). However, as CD1-deficient mice can produce IgE in response to anti-IgD (13, 14, 21), their role in Th2 lineage commitment needs clarification. Furthermore, it is clear that NK1.1+CD4+ T cells are not essential for induction of all forms of Th2 responses. Susceptibility of BALB/c mice to infection with Leishmania major, which is attributed to the development of Th2 cells, is not reversed in β2-microglobulin-deficient mice (22, 23, 24). Also, Th2 responses to other well-characterized Th2 Ags develop normally in these mice (24, 25).

Despite discrepancies concerning their role in directing Th2 development, NK1.1+CD4+ T cells appear pleiotropic by nature, being able to perform multiple functions, many of which are related to immune regulation, suggesting that these cells may play an important central role as immunoregulatory cells, particularly in regard to cell-mediated immune responses. These include cytotoxic activity against viruses (26) and tumors (27), possibly participating in thymic selection through Fas-mediated cytolysis (28), as regulatory cells in autoimmune responses (29, 30) and as inducers of CD8 effector function against intracellular infections (31).

Although all NK1.1+CD4+ T cells are TCRint,3 they represent only a subpopulation of TCRint T cells (32). Analysis of the CD4/8 phenotype of NK1.1TCRint cells indicates that most are CD8 and a few are CD4 (32). Currently, the function of NK1.1TCRint T cells is unknown, in particular, whether they can rapidly produce high concentrations of IL-4 and IFN-γ upon primary CD3 stimulation. Indeed, it has been widely reported that only T cells expressing the NK1.1 Ag possess this unique capability (5, 8, 15, 17), implying that NK1.1TCRint T cells are unable to perform such a function. In this study, we demonstrate that a NK1.1- and DX5-negative population of CD4+TCRint T cells, defined on the basis of FcγR expression, exists in naive NK1.1 allelic positive and negative mice, with the ability to rapidly produce very high amounts of both IL-4 and IFN-γ protein following primary in vitro CD3 stimulation.

Specific pathogen-free female C3H/HeN and C57BL/6 mice (3–6 mo) were purchased from the National Cancer Institute-Frederick Cancer Research Facility Animal Production Area (Frederick, MD). The animals were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current National Institutes of Health regulations and standards. All animal procedures were approved by the Institutional Animal Care and Use Committee. Within each experiment, the mice were age matched. The mice received National Institute of Health-31 open formula mouse chow and sterile water ad libitum. Ambient light was controlled to provide regular cycles of 12 h of light and 12 h of darkness.

Hamster anti-mouse CD3 (2C11, IgG); rat anti-mouse FcγR, and PE and biotin rat anti-mouse FcγR (2.4G2, IgG2b); Cy-Chrome rat anti-mouse CD4 (RM4-5, IgG2a); FITC hamster anti-mouse TCRαβ (H57-597, IgG); mouse anti-mouse NK1.1 and biotin mouse anti-mouse NK1.1 (PK136, IgG2a); rat anti-mouse pan-NK cells (DX5, IgM); FITC rat anti-mouse CD44 (IM7, IgG2b); biotin mouse anti-mouse 3G11 disialoganglioside Ag (SM3G11, IgM); mouse anti-rat IgM and PE mouse anti-rat IgM (G53-238, IgG1); and streptavidin-FITC Abs were purchased from PharMingen (San Diego, CA). RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 5 or 10% bovine calf serum (BCS; HyClone Laboratories, Logan, UT), 2-ME (50 μM), l-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), nonessential amino acids (1×), vitamins (1×), HEPES (0.01 M), and sodium bicarbonate (7.5%) were used as tissue culture medium (cRPMI).

Using the appropriate Abs, fluorescence staining of 5 × 105 enriched splenic CD4+ T cells or 5 × 104 CD4+ T cell subpopulations was performed at 4°C in 100 μl of PBS containing 0.5% BSA, 1% goat serum, and 0.5% NaN3. Both direct and indirect staining events involved 1-h incubations, followed by extensive washing. Fluorescence analysis was conducted by flow-cytometric analysis using a Coulter Epics Profile analyzer (Coulter, Fullerton, CA). Phenotypic analysis of splenic CD4+ T cells for the expression of FcγR expression involved gating out large granular cells and analyzing only small lymphoid-like cells.

Mice were sacrificed and their spleens were removed, single cell suspensions were prepared, and contaminating erythrocytes were lysed with ammonium chloride. The remaining cells were washed, resuspended in cRPMI containing 5% BCS, and then incubated on nylon wool columns (33) to enrich for T lymphocytes. Cells eluted from the nylon wool columns were then enriched for CD4+ T cells by negative selection. Briefly, cells were resuspended in PBS supplemented with 2% BCS and 5% normal rat serum (NRS) and incubated at 4°C with a mixture of biotinylated Abs to markers expressed on unwanted cells, supplied by the manufacturer (Stem Cell Technologies, Vancouver, Canada). These cells were then magnetically labeled by incubating with bispecific tetrameric Ab complexes and magnetic dextran iron particles. The cell suspension was then passed through a high gradient magnetic column of stainless steel mesh with the magnetically labeled cells binding to the column, while the unlabeled CD4+ T cells passed through. The eluted cells were stained with anti-mouse CD4 and TCRαβ Abs to check for purity. Typically, no more than 2% contaminant cells were observed.

Purified CD4+ T cells (≥98%) from C3H/HeN (NK1.1 allelic negative) or C57BL/6 (NK1.1 allelic positive) mice were resuspended in PBS supplemented with 2% BCS and 5% NRS and stained with a rat anti-mouse pan NK cell (DX5, IgM) Ab for 30 min at 4°C using bidirectional rotation. The cells were then washed three times, resuspended in the same staining buffer, and by rotation stained with a secondary mouse anti-rat IgM (IgG) Ab for 30 min at 4°C. In other experiments, under the same conditions purified CD4+ T cells (≥98%) from C57BL/6 mice were stained with a mouse anti-mouse NK1.1 (IgG) Ab. After three washes, cells from either mouse strain were then resuspended in PBS supplemented with 0.1% BSA, at a concentration of 2 × 107/ml. Sheep anti-mouse IgG (Fc)-conjugated immunomagnetic beads (Dynal, Great Neck, NY) were added at a target cell:bead ratio of 1:4 and incubated at 4°C for 30 min. Dynabeads with labeled cells attached were isolated using a Dynal magnetic particle concentrator. After removing the negative cell population, they were then extensively washed and the beads were detached mechanically from the cells by pipetting them up and down against the walls of a 15-ml polypropylene centrifuge tube (Life Technologies), held against a Dynal magnet. Typically, of the total population of cells enriched on the basis of DX5 or NK1.1 expression, 85–92% were both CD4 and TCRαβ positive. The NK1.1- or DX5-negative population was then washed, resuspended in PBS supplemented with 2% BCS and 5% NRS, and stained with a rat anti-mouse FcγR Ab for 30 min at 4°C using bidirectional rotation. The cells were then washed three times and under the same conditions already described for isolating NK1.1+CD4+ and DX5+CD4+ cells, FcγR+CD4+ cells were isolated using sheep anti-rat IgG (Fc)-conjugated immunomagnetic beads (Dynal). Of the total population of cells enriched on the basis of FcγR expression, 87–90% were both CD4 and TCRαβ positive.

Each well of a 96-well microtiter dish (Costar) was coated with 4 μg of anti-mouse CD3 mAb 2C11 overnight at 4°C in coating buffer (0.1 M Na HCO3, pH 8.2) and washed three times with PBS before use. CD4+ T cells were cultured at a density of 2 or 5 × 105 cells/well in 200 μl cRPMI for 3 or 8 h, with and without anti-CD3 mAb. In contrast, FcγR+CD4+, NK1.1+CD4+, and DX5+CD4+ (NK1.1) T cells were cultured at a density of 2 × 105 cells/well in 200 μl cRPMI for 8 h, with and without anti-CD3 stimulation. Supernatants were harvested at 3 and 8 h and analyzed for cytokine secretion by ELISA.

The capture Abs and biotinylated detecting Abs were purchased from PharMingen and used according to the manufacturer’s instructions in a sandwich ELISA procedure, as reported previously (34). Generally, the limit of detection for IL-4 was between 2 and 10 pg/ml, and IFN-γ, 20–50 pg/ml. The test was considered positive if the absorbance value of the experimental group was at least 3 SD greater than the OD of the negative control.

Following 8 h of anti-CD3 stimulation of T cell subsets, the cells were lysed in 50 μl of concentrated guanidine thiocyanate (Direct Protect Lysate RPA Kit; Ambion, Austin, TX). Lysates were vortexed and stored at −80°C until further processing. After thawing, IL-4 mRNA was directly detected and quantitated using a multiprobe ribonuclease protection assay (PharMingen), according to the manufacturer’s instructions. The amount of IL-4 mRNA made by each CD4+ T cell population relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was quantitated by phosphor imager analysis.

It is believed that cells equivalent to NK1.1+CD4+ T cells exist in NK1.1 allelic negative mouse strains. Since a unique feature of these cells is their ability to rapidly produce large concentrations of cytokines, particularly IL-4 and IFN-γ following anti-CD3 stimulation in vivo (17), we wanted to assess whether purified CD4+ T cells from an NK1.1 allelic negative mouse strain (C3H/HeN) could produce these cytokines very early, following primary CD3 stimulation in vitro. In a representative experiment shown in Fig. 1, CD4+ T cells from C3H/HeN mice produced high concentrations of both IL-4 and IFN-γ after 3 or 8 h of anti-CD3 stimulation in vitro, implying that a subpopulation of CD4+ T cells equivalent to NK.1+CD4+ T cells may exist in this mouse strain. Similarly, the prompt production of these cytokines by purified C57BL/6 CD4+ T cells was also detected following 3 or 8 h of CD3 stimulation. However, much higher amounts of IL-4 and IFN-γ were secreted by CD4+ T cells from C3H/HeN mice compared with those produced by C57BL/6 CD4+ T cells, suggesting strain differences exist in the ability of these cells to rapidly produce high concentrations of cytokines.

FIGURE 1.

Rapid cytokine production by CD4+ T cells from NK1.1 allelic positive and negative mice. CD4+ T cells were enriched from the spleens of C3H/HeN or C57BL/6 mice and added to wells of an anti-CD3-coated plate. After 3 and 8 h, IL-4 and IFN-γ secretion was determined by ELISA. The data presented represent the mean and SD from triplicate cultures.

FIGURE 1.

Rapid cytokine production by CD4+ T cells from NK1.1 allelic positive and negative mice. CD4+ T cells were enriched from the spleens of C3H/HeN or C57BL/6 mice and added to wells of an anti-CD3-coated plate. After 3 and 8 h, IL-4 and IFN-γ secretion was determined by ELISA. The data presented represent the mean and SD from triplicate cultures.

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Before the DX5 Ag was recognized as a possible candidate surface marker for NK1.1+CD4+ T cells in all mouse strains, we had been focusing our attention on trying to identify these cells in NK1.1 allelic negative mice, based on their preferential expression of FcγR, another NK cell marker reported to be expressed by these cells. Indeed, based on the dual staining data presented in Fig. 2, we were able to demonstrate that a minor population of FcγR+CD4+ T cells exists in the spleens of both C3H/HeN and C57BL/6 mice approximately equal in magnitude to NK1.1+CD4+ T cells. Thus, it appeared that we could identify cell populations equivalent to NK1.1+CD4+ T cells in NK1.1 allelic negative and positive mice based on FcγR expression. However, to our surprise, when we addressed by triple staining whether the FcγR+CD4+ T cell population identified in C57BL/6 mice was directly equivalent to the NK1.1+CD4+ T cell population, we found that FcγR+CD4+ T cells were largely NK1.1 negative and that a smaller subpopulation of NK1.1+CD4+ T cells expressed the FcγR molecule on their surface (Fig. 2). Staining with an Ab specific for the DX5 Ag, which is expressed by NK1.1+CD4+ T cells and by a subpopulation of CD4+ T cells in NK1.1 allelic negative mice, we also found that the majority of FcγR+CD4+ T cells from C3H/HeN and C57BL/6 mice are DX5 negative (Fig. 2). In terms of proportion, the minor population of DX5+FcγR+CD4+ cells identified in C3H/HeN mice more or less mirrored the minor NK1.1+FcγR+CD4+ T cell population observed in C57BL/6 mice.

FIGURE 2.

Identification of a FcγR+CD4+ T cell subset in C3H/HeN and C57BL/6 mice that is NK1.1 and DX5 negative. CD4-enriched spleen T cells from C3H/HeN and C57BL/6 mice were triple stained with a mixture of anti-CD4, anti-FcγR, and anti-DX5 or anti-NK1.1 mAbs. The percentages in boxes represent the proportion of brightly stained DX5-, FcγR-, and NK1.1-positive cells among the CD4+ T cells. The percentages without boxes represent the proportion of FcγR+CD4+ T cells that brightly stained for DX5 or NK1.1 molecules and the proportion of DX5+CD4+ T cells that brightly stained for FcγR molecules.

FIGURE 2.

Identification of a FcγR+CD4+ T cell subset in C3H/HeN and C57BL/6 mice that is NK1.1 and DX5 negative. CD4-enriched spleen T cells from C3H/HeN and C57BL/6 mice were triple stained with a mixture of anti-CD4, anti-FcγR, and anti-DX5 or anti-NK1.1 mAbs. The percentages in boxes represent the proportion of brightly stained DX5-, FcγR-, and NK1.1-positive cells among the CD4+ T cells. The percentages without boxes represent the proportion of FcγR+CD4+ T cells that brightly stained for DX5 or NK1.1 molecules and the proportion of DX5+CD4+ T cells that brightly stained for FcγR molecules.

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Based on the staining data, it was clear that FcγR+CD4+ T cells represented a unique subpopulation of CD4+ T cells in vivo, distinct from DX5+CD4+ and NK1.1+CD4+ T cells present in C3H/HeN and C57BL/6 mice, respectively. Whether FcγR+CD4+ T cells were related in any way to NK1.1+CD4+ or DX5+CD4+ T cells phenotypically was not known.

FcγR+CD4+ and DX5+CD4+ T cells from C3H/HeN (NK1.1 allelic negative) mice or FcγR+CD4+ and NK1.1+CD4+ T cells from C57BL/6 (NK1.1 allelic positive) mice were positively selected. In some experiments, CD4+ T cells from C57BL/6 mice were enriched on the basis of DX5 expression so that a phenotypic comparison could be made between this population and CD4+ T cells enriched on the basis of NK1.1 expression. It is important to point out that within each experiment, the relative proportion of each CD4+ T cell subset isolated reflected their staining profile in vivo. In other words, regardless of the mouse strain used, almost three times as many DX5+CD4+ T cells were isolated compared with FcγR+CD4+ and NK1.1+CD4+ T cells. Each CD4+ subpopulation was analyzed for levels of TCRαβ expression and activation versus naive status. As illustrated in Fig. 3, FcγR+CD4+ T cells isolated from C3H/HeN or C57BL/6 mice were TCRint, mirroring the levels expressed by NK1.1+CD4+ and DX5+CD4+ T cells isolated from the same mice. Thus, it appeared that the CD4+ T cells that preferentially expressed FcγR molecules on their surface represented a subset of TCRint cells. Further evidence that FcγR+CD4+ T cells may be related to DX5+CD4+ T cells is presented in Fig. 4. An analysis of the activation status of FcγR+CD4+ and DX5+CD4+ T cells isolated from C3H/HeN mice revealed that they stained CD44high and 3G11high (Fig. 4). Similarly, NK1.1+CD4+ isolated from C57BL/6 mice exhibited the same phenotype (data not shown). In contrast, FcγRCD4+ and DX5CD4+ T cells were CD44low and 3G11high.

FIGURE 3.

Intermediate levels of TCRαβ expression by FcγR+CD4+ and NK1.1/DX5+CD4+ splenic T cells isolated from NK1.1 allelic positive and negative mice. Conventional CD4+ T cells were enriched from the spleens of C3H/HeN or C57BL/6 mice and stained with FITC anti-TCRαβ mAb (grey lines). FcγR+CD4+ and NK1.1/DX5+CD4+ populations were selected by magnetic bead enrichment and stained with FITC anti-TCRαβ mAb (black lines).

FIGURE 3.

Intermediate levels of TCRαβ expression by FcγR+CD4+ and NK1.1/DX5+CD4+ splenic T cells isolated from NK1.1 allelic positive and negative mice. Conventional CD4+ T cells were enriched from the spleens of C3H/HeN or C57BL/6 mice and stained with FITC anti-TCRαβ mAb (grey lines). FcγR+CD4+ and NK1.1/DX5+CD4+ populations were selected by magnetic bead enrichment and stained with FITC anti-TCRαβ mAb (black lines).

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FIGURE 4.

Activation status of splenic CD4+ T cell subpopulations isolated from C3H/HeN mice. CD4+ T cell subpopulations were dual stained with Cy-Chrome anti-CD4 and FITC anti-CD44 or biotin anti-3G11 and streptavidin FITC mAbs. The percentages shown represent the proportion of CD4+ T cells within each subpopulation, which stained bright and dull for CD44/3G11.

FIGURE 4.

Activation status of splenic CD4+ T cell subpopulations isolated from C3H/HeN mice. CD4+ T cell subpopulations were dual stained with Cy-Chrome anti-CD4 and FITC anti-CD44 or biotin anti-3G11 and streptavidin FITC mAbs. The percentages shown represent the proportion of CD4+ T cells within each subpopulation, which stained bright and dull for CD44/3G11.

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Although all NK1.1+CD4+ T cells are TCRint, they represent only a subpopulation of CD4+TCRint cells. To date, whether other NK1.1CD4+TCRint subsets share the unique capability of being able to rapidly secrete high amounts of cytokines such as IFN-γ and IL-4 is not known. Since we were able to isolate NK1.1DX5CD4+TCRint T cells on the basis of FcγR expression and NK1.1/DX5+CD4+ T cells from the same C3H/HeN or C57BL/6 mouse strain, we wanted to compare these CD4+TCRint T cell subsets in terms of their ability to rapidly secrete high amounts of IL-4 and IFN-γ upon CD3 stimulation in vitro. At the same time, we also examined the IL-4 mRNA levels produced by DX5+CD4+ and FcγR+CD4+ T cells isolated from C3H/HeN mice. As mentioned above, a very small population of NK1.1+FcγR+CD4+ or DX5+FcγR+CD4+ T cells exists in C57BL/6 and C3H/HeN mice, respectively. Therefore, if FcγR+CD4+ T cells were isolated first, NK1.1+FcγR+CD4+ or DX5+FcγR+CD4+ T cells would also be enriched, thus preventing us from being able to fully characterize the FcγR+CD4+ T cell population in terms of function. So, to reduce the chances of this happening, we first enriched for NK1.1+CD4+ or DX5+CD4+ T cells and then FcγR+CD4+ T cells. When the CD4+ T cell subsets were isolated in this order, no more than 2% of FcγR+CD4+ T cells stained positive for NK1.1 or DX5 expression, representing a contamination of about 3000-4000 NK1.1+FcγR+CD4+ or DX5+FcγR+CD4+ T cells of the 2 × 105 cells (of which 87–90% were TCRαβ+) added to each anti-CD3-coated well. As shown in Fig. 5, following 8 h of primary CD3 stimulation, both DX5/NK1.1+CD4+ and FcγR+CD4+ T cells isolated from C3H/HeN or C57BL/6 mice rapidly produced substantially high amounts of IL-4 protein. Furthermore, it appeared that DX5/NK1.1+CD4+ T cells produced twice as much IL-4 as FcγR+CD4+ T cells. Interestingly, despite differences in the levels of IL-4 protein produced by these CD4+ T cell subpopulations, FcγR+CD4+ T cells selected from C3H/HeN mice made more IL-4 mRNA than DX5+CD4+ T cells, isolated from the same mice (Fig. 6). In contrast, the high levels of IFN-γ detected upon anti-CD3 stimulation of FcγR+CD4+ T cells from C3H/HeN mice were not measurable if the same population was isolated from C57BL/6 mice and treated under exactly the same conditions. To support our finding that the high amounts of IL-4 and IFN-γ protein were indeed being produced by the FcγR+CD4+ T cell population and not the 2% contaminating population, a titration of DX5+FcγR+/−CD4+ or NK1.1+FcγR+/−CD4+ T cells from C3H/HeN and C57BL/6 mice, respectively, was conducted. To each well of an anti-CD3-coated plate, 10,000, 20,000, or 30,000 NK1.1+FcγR+/−CD4+ or DX5+FcγR+/−CD4+TCRαβ+ T cells were added, representing a contamination of at least 6, 11, and 17%, respectively. We found that at these cell concentrations no IL-4 or IFN-γ protein could be detected, implying that the source of the IL-4 and IFN-γ protein measured in previous experiments was the FcγR+CD4+ T cell population and could not be attributed to the small amount of contaminating cells.

FIGURE 5.

FcγR+CD4+ and NK1.1/DX5+CD4+ splenic T cells isolated from NK1.1 allelic positive and negative mice rapidly produced large amounts of cytokines upon CD3 stimulation. FcγR+CD4+, NK1.1/DX5+CD4+, DX5/NK1.1FcγRCD4+, and CD4+ T cells were cultured in anti-CD3-coated wells at a concentration of 2 × 105 cells/well for 8 h. Cytokine production was analyzed by ELISA. The mean and SD from triplicate cultures are presented.

FIGURE 5.

FcγR+CD4+ and NK1.1/DX5+CD4+ splenic T cells isolated from NK1.1 allelic positive and negative mice rapidly produced large amounts of cytokines upon CD3 stimulation. FcγR+CD4+, NK1.1/DX5+CD4+, DX5/NK1.1FcγRCD4+, and CD4+ T cells were cultured in anti-CD3-coated wells at a concentration of 2 × 105 cells/well for 8 h. Cytokine production was analyzed by ELISA. The mean and SD from triplicate cultures are presented.

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FIGURE 6.

FcγR+CD4+ splenic T cells made more IL-4 mRNA than DX5+CD4+ splenic T cells isolated from the same C3H/HeN mouse strain, upon CD3 stimulation. FcγR+CD4+, DX5+CD4+, DX5FcγRCD4+, and CD4+ splenic T cells were enriched from the spleens of C3H/HeN mice and were cultured in anti-CD3-coated dishes. The cells were then harvested and lysed, and IL-4 mRNA was detected, using a multiprobe ribonuclease protection assay. The amount of IL-4 mRNA made by each CD4+ T cell population relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was quantitated by phosphor imager analysis. The signal intensity is presented as relative pixel density (lower).

FIGURE 6.

FcγR+CD4+ splenic T cells made more IL-4 mRNA than DX5+CD4+ splenic T cells isolated from the same C3H/HeN mouse strain, upon CD3 stimulation. FcγR+CD4+, DX5+CD4+, DX5FcγRCD4+, and CD4+ splenic T cells were enriched from the spleens of C3H/HeN mice and were cultured in anti-CD3-coated dishes. The cells were then harvested and lysed, and IL-4 mRNA was detected, using a multiprobe ribonuclease protection assay. The amount of IL-4 mRNA made by each CD4+ T cell population relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was quantitated by phosphor imager analysis. The signal intensity is presented as relative pixel density (lower).

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All NK1.1+CD4+ T cells are TCRint, possess an activated surface phenotype, and are able to rapidly produce very high amounts of cytokines, notably IL-4, upon primary TCR stimulation. Studies addressing the functional potential of CD4+NK1.1+ T cells in vitro and in vivo, by triggering their TCR-CD3 complex, suggested that virtually all of the IL-4 was produced by them, and not NK1.1 T cells (15, 17). Although all NK1.1+CD4+ T cells are TCRint, other subsets of NK1.1CD4+TCRint T cells exist (32). It is therefore inferred, based on these studies, that TCRint subsets not expressing NK1.1 would not rapidly secrete large amounts of IL-4 upon CD3 stimulation. We show in this study, however, that splenic NK1.1CD4+TCRint T cells, selected on the basis of FcγR expression, isolated from NK1.1 allelic positive or negative mice, can rapidly produce high quantities of IL-4 upon primary CD3 stimulation in vitro. Although NK1.1+CD4+ T cells appeared to be the source of all of the early IL-4 detected, Yoshimoto and Paul (17) still found by RT-PCR low levels of IL-4 mRNA in the much larger NK1.1 T cell population, suggesting that an as yet unidentified population of cells may exist with a similar function. Since the NK1.1 T cell population we identified appears related to NK1.1+CD4+ T cells, in terms of phenotype and function and is equally small, it seems likely that we have identified such a population.

We also report in this work that DX5+CD4+ T cells isolated from NK1.1 allelic negative mice are able to rapidly produce very large quantities of IL-4 and IFN-γ, upon anti-CD3 stimulation in vitro. Since these cells are TCRint and exhibit an activated phenotype, it appears that the DX5 Ag is a good marker for distinguishing an equivalent population of NK1.1+CD4+ T cells in NK1.1 allelic negative mice. In this regard, it is worth mentioning that DX5+CD4+ T cells, isolated from C57BL/6 mice, produced more or less the same level of early IL-4 as NK1.1+CD4+ T cells (data not shown).

Based on our findings, at least three different subsets of TCRintCD4+ T cells exist in the spleens of naive pathogen-free mice, each one expressing one or more surface markers usually expressed by NK cells. Whether the three populations observed are each unique in terms of function or represent the same population, exhibiting different stages of activation, is not known. Certainly, the finding that FcγR+CD4+ T cells produced different amounts of IL-4 mRNA and protein compared with DX5/NK1.1+CD4+ T cells, in response to CD3 stimulation, would support either notion.

It is clear that NK1.1+CD4+ T cells can release both IL-4 and IFN-γ when stimulated through their TCR (16). However, these cells can also differentially release these cytokines depending on how they are stimulated. Triggering them with CD1 induced substantial amounts of IL-4, but very little IFN-γ (16), whereas cross-linking NK1.1 molecules induced IFN-γ release, but not IL-4 production (35), suggesting that under physiologic conditions, the production of IL-4 and IFN-γ can be differentially regulated through the engagement of different surface receptors. Unlike the NK1.1 molecule, which is expressed by NK cells and a subpopulation of T cells, the FcγR is the most broadly distributed FcR, being found on cells of every hemopoietic lineage (36). Although the function of the FcγR molecule in activating NK1.1+CD4+ T cells is unknown, it is clear that a diversity of important functions is linked to this molecule, including Ab-dependent cellular cytotoxicity, Ab-mediated feedback inhibition, and the triggering of cytokine and superoxide production by mononuclear phagocytes and lymphocytes (36). Thus, CD4+TCRint T cell subpopulations that express this molecule may possess distinct functional capabilities different from those of DX5/NK1.1+FcγRCD4+ T cells. Therefore, if FcγR+CD4+, DX5/NK1.1+CD4+, and DX5/NK1.1+FcγR+CD4+ T cells do indeed represent different cell populations (as suggested by the data presented in this work), then the possibility emerges that TCRintCD4+ T cells may represent a unique family of immunoregulatory T cells, each member having the capacity to direct the immune response, by releasing different cytokines depending on the nature of the signal that activates the cell.

When NK1.1+CD4+ T cells are activated by CD3 stimulation in vitro, they no longer express NK1.1 molecules on their surface, but still maintain their ability to secrete high quantities of IL-4 (37). Using common γ-chain-deficient mice, it was found that NK thymocytes that failed to coexpress the NK-associated marker NKR-P1 could produce normal amounts of IL-4 (38), implying that the IL-4-producing phenotype is not dependent on the acquisition of NK-associated markers. Conversely, it is also well documented that resting adult T cells do not express FcR molecules, but on TCR-triggered activation, Fc receptor induction occurs both in vitro and in vivo (36). Therefore, as NK1.1+ T cells appear more closely related to T cells than NK cells (38), the possibility arises that NK1.1/DX5FcγR+CD4+ T cells may represent a population of activated NK1.1/DX5+CD4+ T cells in vivo, while NK1.1/DX5+FcγR+CD4+ T cells represent a population in the transition of becoming fully activated. On the other hand, the NK1.1/DX5+FcγRCD4+ T cell population observed in vivo may represent an unprimed population.

Although it has been widely reported that NK1.1+ T cells exhibit an activated phenotype, expressing high levels of CD44 and low levels of 3G11 (5, 8), to the best of our knowledge 3G11 expression has only been examined on thymic NK1.1+ T cells, and not splenic CD4+NK1.1+ cells. This may explain the discrepancy between our findings with splenic cells and those reported in the literature. It is also worth mentioning that when using the same cell surface markers to address the activation status of conventional T cells and NK1.1+CD4+ T cells a paradox appears to emerge. Conventional T cells that exist in naive germfree animals and therefore have not previously encountered an Ag, exhibit a naive phenotype, whereas NK1.1+CD4+ T cells in the same mice display an activated state, suggesting that a truly naive cell can still express activation markers. This raises the question as to whether or not we can address the activation status of NK1.1+CD4+ T cells using the cell surface markers traditionally used to characterize conventional T cells. Our finding that NK1.1/DX5+CD4+ and FcγR+CD4+ T cells express high levels of 3G11, a molecule expressed at low levels on activated conventional CD4+ T cells, would suggest that this is not the case.

A unique feature of splenic NK1.1+CD4+ T cells is their ability to rapidly produce high quantities of cytokines, upon in vivo stimulation with anti-CD3 mAb (17). Spleen cells harvested from mice injected with anti-CD3 mAb 90 min earlier were found to produce large amounts of IL-4 after 1 h of culture (17). In contrast, if the same concentration of spleen cells, taken from uninjected mice, was stimulated in vitro with plate-bound CD3 mAb, substantial amounts of IL-4 were not detected until 24 h of culture. Furthermore, these cells produced approximately one-half of the amount of IL-4 secreted by in vivo stimulated spleen cells cultured for 1 h (17). Studies examining the cytokine-secreting potential of FACS-sorted splenic NK1.1+CD4+ T cells upon in vitro CD3 stimulation also reported detecting IL-4 protein after 24 h of culture, but not earlier (16). Currently, the difference between in vivo and in vitro responses to anti-CD3 is unexplained. One proposal is that some critical, as yet unidentified, element of the in vivo architecture is responsible for speeding up cytokine secretion (1). In contrast to previous findings, we report in this work, however, that purified splenic NK1.1+CD4+ T cells stimulated with plate-bound anti-CD3 mAb for 8 h produced large quantities of IL-4 and IFN-γ. Thus, it appears that splenic NK1.1+CD4+ T cells are able to rapidly secrete cytokines upon stimulation in vitro, as observed in vivo. In general, the early production of high amounts of IL-4 and IFN-γ could only be detected, however, if NK1.1+CD4+ T cells were cultured at ≥105 cells/well (data not shown). Although not disputing that the in vivo architecture may influence the ability of NK1.1+CD4+ T cells to rapidly secrete cytokines, it is also conceivable that another reason investigators are not able to detect very early levels of cytokines upon CD3 stimulation in vitro is because of the very low cell numbers of NK1.1+CD4+ T cells (104) seeded into a culture plate well (16). Since FACS sorting such a small T cell subset from purified splenic CD4+ T cells gives a very low cell yield, investigators are restricted in terms of the number of cells they can stimulate. In contrast, by positively selecting NK1.1+CD4+ T cells directly from a very large population of purified splenic CD4+ T cells, we were able to obtain a relatively high yield of cells, enabling us to activate up to 2 × 105 cells/well.

Interestingly, although IFN-γ was detected after culturing 2 × 105 NK1.1+CD4+ T cells with anti-CD3 mAb, no IFN-γ was detected when FcγR+CD4+ T cells from C57BL/6 mice were stimulated at the same concentration. Since FcγR+CD4+ T cells isolated from NK1.1 allelic negative mice rapidly produced high quantities of IFN-γ, it seems unlikely that the same cells from NK1.1 allelic positive mice would not be able to perform this function. Indeed, we found that under the same conditions of stimulation, DX5+CD4+ and FcγR+CD4+ cells from C3H/HeN mice produced approximately eightfold higher levels of cytokines compared with CD4+ subpopulations isolated from C57BL/6 mice, implying strain variation. Thus, a more plausible explanation is that more FcγR+CD4+ T cells from C57BL/6 mice must be stimulated in order for early IFN-γ to be detected, in contrast to the same population isolated from C3H/HeN mice.

Whether NK1.1/DX5+CD4+ and FcγR+CD4+ T cell subsets are unique populations of cells, each capable of performing a different set of functions, related to the complexity of their surface receptors or whether they represent different activation states of the same population, is certainly intriguing and difficult to address. Regardless, they all share two features in common. They express intermediate levels of TCRαβ molecules on their surface and possess the capacity to rapidly produce large quantities of cytokines, notably IL-4 and IFN-γ upon TCR stimulation. Therefore, perhaps we should consider NK1.1/DX5+CD4+ and FcγR+CD4+ T cells as being members of a larger population of CD4+TCRint immunoregulatory T cells.

We thank Karen Ramirez for skilled operation of the Coulter Epics Profile analyzer and Todd Giese who conducted the ELISAs. We also thank Vijay Shreedhar for helpful discussions and Jeff Walterscheid for help in preparing the manuscript.

1

This work was supported by grants from the National Institutes of Health (ES 07327 and CA 75575). The animal facilities at M. D. Anderson Cancer Center are supported in part by a Core Grant from the National Cancer Institute (CA 16672).

3

Abbreviations used in this paper: TCRint, intermediate levels of TCRαβ expression; BCS, bovine calf serum; cRPMI, complete medium containing supplements and antibiotics; NRS, normal rat serum.

1
Bendelac, A., M. N. Rivera, S.-H. Park, J. H. Roark.
1997
. Mouse CD1-specific NK1 T cells: development, specificity, and function.
Annu. Rev. Immunol.
15
:
535
2
Arase, H., N. Arase, K. Ogasawara, R. A. Good, K. Onoe.
1992
. An NK1.1+CD4+CD8 single-positive thymocyte subpopulation that expresses a highly skewed T-cell antigen receptor Vβ family.
Proc. Natl. Acad. Sci. USA
89
:
6506
3
Ohteki, T., H. R. MacDonald.
1994
. Major histocompatibility complex class I related molecules control the development of CD4+CD8 and CD4CD8 subsets of natural killer 1.1+ T cell receptor-α/β+ cells in the liver of mice.
J. Exp. Med.
180
:
699
4
Sykes, M..
1990
. Unusual T cell populations in adult murine bone marrow: prevalence of CD3+CD4CD8 and αβTCR+NK1.1+ cells.
J. Immunol.
145
:
3209
5
Vicari, A. P., A. Zlotnik.
1996
. Mouse NK1.1+ T cells: a new family of T cells.
Immunol. Today
17
:
71
6
Lantz, O., A. Bendelac.
1994
. An invariant T cell receptor α chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4CD8 T cells in mice and humans.
J. Exp. Med.
180
:
1097
7
Koseki, H., H. Asano, T. Inaba, N. Miyashita, K. Moriwaki, K. Fischer Lindahl, Y. Mizutani, K. Imai, M. Taniguchi.
1991
. Dominant expression of a distinctive V14+ T-cell antigen receptor α chain in mice.
Proc. Natl. Acad. Sci. USA
88
:
7518
8
MacDonald, H. R..
1995
. NK1.1+ T cell receptor-α/β+ cells: new clues to their origin, specificity, and function.
J. Exp. Med.
182
:
633
9
Ortaldo, J. R., R. Winkler-Pickett, A. T. Mason, L. H. Mason.
1998
. The Ly-49 family: regulation of cytotoxicity and cytokine production in murine CD3+ cells.
J. Immunol.
160
:
1158
10
Bendelac, A., N. Killeen, D. R. Littman, R. H. Schwartz.
1994
. A subset of CD4+ thymocytes selected by MHC class I molecules.
Science
263
:
1774
11
Sato, K., K. Ohtsuka, K. Hasegawa, S. Yamagiwa, H. Watanabe, H. Asakura, T. Abo.
1995
. Evidence for extrathymic generation of intermediate T cell receptor cells in the liver revealed in thymectomized, irradiated mice subjected to bone marrow transplantation.
J. Exp. Med.
182
:
759
12
Bendelac, A., O. Lantz, M. E. Quimby, J. W. Yewdell, J. R. Bennink, R. R. Brutkiewicz.
1995
. CD1 recognition by mouse NK1+ T lymphocytes.
Science
268
:
863
13
Chen, Y.-H., N. M. Chiu, M. Mandal, N. Wang, C.-R. Wang.
1997
. Impaired NK1.1+ T cell development and early IL-4 production in CD1-deficient mice.
Immunity
6
:
459
14
Mendiratta, S. K., W. D. Martin, S. Hong, A. Boesteanu, S. Joyce, L. Van Kaer.
1997
. CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4.
Immunity
6
:
469
15
Arase, H., N. Arase, K. Nakagawa, R. A. Good, K. Onoe.
1993
. NK1.1+CD4+CD8 thymocytes with specific lymphokine secretion.
Eur. J. Immunol.
23
:
307
16
Chen, H., W. E. Paul.
1997
. Cultured NK1.1+CD4+ T cells produce large amounts of IL-4 and IFN-γ upon activation by anti-CD3 or CD1.
J. Immunol.
159
:
2240
17
Yoshimoto, T., W. E. Paul.
1994
. CD4pos, NK1.1pos T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3.
J. Exp. Med.
179
:
1285
18
Yoshimoto, T., A. Bendelac, C. Watson, J. Hu-Li, W. E. Paul.
1996
. Role of NK1.1+ T cells in a TH2 response and in immunoglobulin E production.
Science
270
:
1845
19
Yoshimoto, T., A. Bendelac, J. Hu-Li, W. E. Paul.
1995
. Defective IgE production by SJL mice is linked to the absence of CD4+, NK1.1+ T cells that promptly produce interleukin 4.
Proc. Natl. Acad. Sci. USA
92
:
11931
20
Bendelac, A., R. D. Hunziker, O. Lantz.
1996
. Increased interleukin 4 and immunoglobulin E production in transgenic mice overexpressing NK1 T cells.
J. Exp. Med.
184
:
1285
21
Smiley, S. T., M. H. Kaplan, M. J. Grusby.
1997
. Immunoglobulin E production in the absence of interleukin-4-secreting CD1-dependent cells.
Science
275
:
977
22
Launois, P., T. Ohteki, K. Swihart, H. R. MacDonald, J. A. Louis.
1995
. In susceptible mice, Leishmania major induce very rapid interleukin-4 production by CD4+ T cells which are NK1.1.
Eur. J. Immunol.
25
:
3298
23
Von der Weid, T., A. M. Beebe, D. C. Roopenian, R. L. Coffman.
1996
. Early production of IL-4 and induction of Th2 responses in the lymph node originate from an MHC class I-independent CD4+NK1.1 T cell population.
J. Immunol.
157
:
4421
24
Brown, D. R., D. J. Fowell, D. B. Corry, T. A. Wynn, N. H. Moskowitz, A. W. Cheever, R. M. Locksley, S. L. Reiner.
1996
. β2-microglobulin-dependent NK1.1+ T cells are not essential for T helper cell 2 immune responses.
J. Exp. Med.
184
:
1295
25
Zhang, Y., K. H. Rogers, D. B. Lewis.
1996
. β2-microglobulin-dependent T cells are dispensable for allergen-induced T helper 2 responses.
J. Exp. Med.
184
:
1507
26
Yang, Y., J. M. Wilson.
1995
. Clearance of adenovirus-infected hepatocytes by MHC class I-restricted CD4+CTLs in vivo.
J. Immunol.
155
:
2564
27
Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki, M. Kanno, M. Taniguchi.
1997
. Requirement for Vα14 NKT cells in IL-12-mediated rejection of tumors.
Science
278
:
1623
28
Arase, H., N. Arase, Y. Kobayashi, Y. Nishimura, S. Yonehara, K. Onoe.
1994
. Cytotoxicity of fresh NK1.1+ T cell receptor α/β+ thymocytes against a CD4+CD8+ thymocyte population associated with intact Fas antigen expression on the target.
J. Exp. Med.
180
:
423
29
Takeda, K., G. Dennert.
1993
. The development of autoimmunity in C57BL/6 lpr mice correlates with the disappearance of natural killer type 1-positive cells: evidence for their suppressive action on bone marrow stem cell proliferation, B cell immunoglobulin secretion, and autoimmune symptoms.
J. Exp. Med.
177
:
155
30
Mieza, M. A., T. Itoh, J. Q. Cui, Y. Makino, T. Kawano, K. Tsuchida, T. Koike, T. Shirai, H. Yagita, A. Matsuzawa, H. Koseki, M. Taniguchi.
1996
. Selective reduction of Vα14+ NK T cells associated with disease development in autoimmune-prone mice.
J. Immunol.
156
:
4035
31
Denkers, E. Y., T. Scharton-Kersten, S. Barbieri, P. Caspar, A. Sher.
1996
. A role for CD4+NK1.1+ T lymphocytes as major histocompatibility complex class II independent helper cells in the generation of CD8+ effector function against intracellular infection.
J. Exp. Med.
184
:
131
32
Watanabe, H., C. Miyaji, Y. Kawachi, T. Iiai, K. Ohtsuka, T. Iwanage, H. Takahashi-Iwanaga, T. Abo.
1995
. Relationships between intermediate TCR cells and NK1.1+ T cells in various immune organs: NK1.1+ T cells are present within a population of intermediate TCR cells.
J. Immunol.
155
:
2972
33
Julius, M. H., E. Simpson, L. A. Herzenberg.
1973
. A rapid method for the isolation of functional thymus-derived murine lymphocytes.
Eur. J. Immunol.
3
:
645
34
Shreedhar, V., T. Giese, V. W. Sung, S. E. Ullrich.
1998
. A cytokine cascade including prostaglandin E2, IL-4, and IL-10 is responsible for UV-induced systemic immune suppression.
J. Immunol.
160
:
3783
35
Arase, H., N. Arase, T. Saito.
1996
. Interferon γ production by natural killer (NK) cells and NK1.1+ T cells upon NKR-P1 cross-linking.
J. Exp. Med.
183
:
2391
36
Lynch, R. G..
1998
. The biology and pathology of lymphocyte Fc receptors.
Am. J. Pathol.
152
:
631
37
Chen, H., H. Huang, W. E. Paul.
1997
. NK1.1+CD4+ T cells lose NK1.1 expression upon in vitro activation.
J. Immunol.
158
:
5112
38
Lantz, O., L. I. Sharara, F. Tilloy, A. Andersson, J. P. DiSanto.
1997
. Lineage relationships and differentiation of natural killer (NK) T cells: intrathymic selection and interleukin (IL)-4 production in the absence of NKR-P1 and Ly49 molecules.
J. Exp. Med.
185
:
1395