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.     

Origin and differentiation of natural killer cells. II. Functional and morphologic studies of purified NK-1.1+ cells

Cells bearing the NK-specific marker NK-1.1 were purified from mouse spleens by utilizing a monoclonal anti-NK-1.1 antibody and cell sorting. In normal adult mice, all of the splenic NK activity against YAC-1 cells was found in the NK-1.1+ fraction, whereas NK-1.1- cells were depleted of NK activity. The NK activity of sorted NK-1.1+ cells was enriched 15- to 30-fold over unfractionated spleen cells. Light and electron microscopic studies of purified NK-1.1+ cells showed a homogeneous population of cells, each containing one to four cytoplasmic granules. Mice whose bone marrow has been destroyed by chronic exposure to 17-beta-estradiol have very low NK activity. However, spleen cells of estradiol-treated mice contained a normal frequency of NK-1.1+ cells which bound to YAC-1 cells, but failed to lyse them even after purification and subsequent exposure to interferon-alpha/beta in vitro. It appears, therefore, that in the absence of intact bone marrow, NK-1.1+ cells may be arrested in a nonlytic and interferon-unresponsive state. Spleens of neonatal mice which have low NK activity were analyzed to ascertain whether immature NK-1.1+ cells, similar to those found in estradiol-treated mice, could be demonstrated. Spleens of 8- to 9-day-old mice also contained NK-1.1+ cells which had very low NK activity even after purification. Sorted NK-1.1+ cells were examined for cytotoxicity in mice whose NK activity was suppressed by pretreatment with Corynebacterium parvum (-15 days). In contrast to cells from estradiol-treated and neonatal mice, NK-1.1+ from mice treated with C. parvum had normal functional activity. Similarly, although NK activity of unfractionated bone marrow cells is low, sorted NK-1.1+ cells were greatly enriched for lytic activity. Thus, we conclude that cell sorting with monoclonal anti-NK-1.1 antibody provides a powerful tool for examining the mechanisms underlying various states of low NK activity, and there exist NK-1.1+, nonlytic, interferon-unresponsive cells which apparently require an intact marrow microenvironment for differentiation into mature, lytic NK cells.     

Effector and precursor phenotypes of lymphokine-activated killer cells in mice with severe combined immunodeficiency (scid) and athymic (nude) mice

The lineage of lymphokine-activated killer (LAK) cells is poorly understood. To examine the relationship between LAK and natural killer (NK) cells we utilized two congenitally immunodeficient mice, namely severe combined immunodeficient (scid) and athymic (nude) mice that lack T cells but have normal NK cells. LAK activity was evaluated by the ability to lyze NK-resistant P815 cells. When cultured with human recombinant interleukin 2, splenocytes of scid and nude mice could generate LAK activity at levels comparable to or more than those of normal C.B-17 mice. LAK effector cells in these immunodeficient mice as well as normal mice had the phenotype resembling that of NK cells with asialo-GM1 (aGM1) expression. In vivo treatment with anti-aGM1 antiserum completely abolished the induction of LAK activity from splenocytes of normal mice. In contrast, LAK activity in splenocytes of scid and nude mice was still demonstrable even after this treatment, indicating that most LAK precursors in both mice were cells without aGM1 antigen. The aGM1- progenitors for LAK activity, probably in common with NK progenitors, appeared to be more expanded in scid and nude mice than in normal mice. The use of such congenitally immunodeficient mice should be helpful in studying the differentiation step of LAK as well as NK cells from their precursors.     

T cell receptor delta gene organization and expression in normal and leukemic natural killer cells

In peripheral blood most NK activity is mediated by CD3- cells with large granular lymphocyte morphology which cannot be assigned to a specific hemopoietic lineage. In accordance with previous studies we have analyzed the organization of the TCR delta gene, which rearranges early in thymic ontogeny, in normal NK cells, and in granular lymphocytes proliferative disorders (GLPD), in an effort to further define their relationship to the T cell differentiation pathway and to identify a possible marker of clonality for CD3- GLPD. The alpha/delta locus was rearranged in five cases of CD3+ GLPD with a biallelic deletion of the C delta region, suggesting V-J alpha rearrangement, whereas CD3- GLPD and normal CD3- NK cells had the delta gene in germ-line configuration, but surprisingly expressed high levels of TCR delta-related mRNA. On the basis of this finding and of the presence of truncated TCR-beta and CD3-epsilon mRNA, we are led to speculate on a possible ontogenic relationship of NK cells to the T cell differentiation pathway at stages preceding TCR gene rearrangement.     

Physiology of natural killer cells. In vivo regulation of progenitors by interleukin 3

Adoptive transfer of bone marrow cells to syngeneic lethally irradiated C57BL/6 mice was used to study the maturation of natural killer (NK) cells from their progenitors. The NK progenitor cell was found to be asialomonoganglioside-negative, (aGM1-) Thy-1-, NK-1-, Ly-1-, Ly-2-, and L3T4-. The NK cells emerging from the bone marrow grafts were aGM1+, NK-1+, Thy-1+/-, Ly-1-, Ly-2-, and L3T4- and to have a target specter similar to that of NK cells isolated from the spleen of normal mice. The regulatory role of interleukin 2 (IL-2) and interleukin 3 (IL-3) for the maturation of NK cells was examined by exposure of the bone marrow cells to the lymphokines in vitro before bone marrow grafting or by treatment of bone marrow-grafted mice with lymphokines through s.c. implanted miniosmotic pumps. IL-3 antagonized the IL-2-induced maturation of NK cells in vitro and strongly inhibited the generation of NK cells after adoptive transfer of bone marrow cells in vivo. The suppressive effect of IL-3 was evident throughout the treatment period (8 or 16 days) but was apparently reversible because NK activity returned to control levels within 8 days after cessation of treatment. The inhibition of cytotoxic activity was accompanied by a reduced appearance of cells with the NK phenotypic markers aGM1 or NK-1, indicating that not only the cytotoxic activity of NK cells but also their actual formation was inhibited. Concomitantly, a moderate increase in cells expressing the T cell marker L3T4 and an increased proliferative response to the T cell mitogen concanavalin A was observed. A direct estimate of the effect of IL-3 on the frequency of NK cell progenitors was obtained by limiting dilution analysis of bone marrow cells at day 8 after bone marrow transplantation. The estimated minimal frequency of NK cell progenitors was reduced from 1/11,800 in control to 1/41,900 in IL-3-exposed mice. IL-3 may take part in the homeostasis of NK cells by the down-regulation of their progenitors.     

The NK-1.1(-) mouse: a model to study differentiation of murine NK cells

The NK-1.1(-) mouse was constructed by weekly injections of monoclonal anti-NK-1.1 antibody from birth through adulthood. Spleen cells from these mice have decreased NK-1.1+ cells and null (Thy-1- and B220-) cells. Their splenic NK activity to YAC targets was low and was not enhanced by IFN-alpha or IFN-beta. Bone marrow (BM) of these NK-1.1(-) mice have normal precursors to NK cells: 1) NK activity could be generated from NK-1.1(-) BM cells cultured in rIL 2 for 5 to 6 days. These cultured BM cells expressed Qa-5, Thy-1, AsGm-1, and NK-1.1 antigens. The precursor cells of these BM cytotoxic cells are NK-1.1-; 2) transfer of BM cells from the NK-1.1(-) mice reconstituted the NK activity of irradiated, NK-depleted recipients. Lymphokine-activated killer cells could also be generated from spleens of these NK-1.1(-) mice. Therefore, the NK-1.1(-) mice were specifically depleted of mature cytotoxic NK cells, but not the NK-1.1- precursors of NK cells. This mouse model is valuable to study ontogeny and physiologic relevance of NK cells.     

Murine NK cell heterogeneity: subpopulations of C57BL/6 splenic NK cells detected by NK-1.1 and NK-2.1 antisera

NK-1.1 antiserum - (BALB/c X C3H)F1 anti-CE - and NK-2.1 antiserum - NZB anti-BALB/c - detect genetically distinct alloantigens on C57BL/6 natural killer (NK) cells. We have analyzed whether these two alloantigens are associated with functional subsets of NK cells. For this study, nylon wool nonadherent C57BL/6 spleen cells (SC) were treated with complement (C) and NK-1.1 or NK-2.1 antisera and then tested for NK activity against a panel of tumor targets in 6- and 19-hour 51Cr release assays. The NK activity against the prototype NK target YAC-1 was reduced equally by both antisera. Similar reductions by both antisera were also observed when SC were tested against another murine lymphoma target, L5178c127v, against the C57BL/6 melanoma B16, and against the human liver cell line Chang. In contrast, NK activity to the lymphoma FBL-3 and the human erythroleukemia K562 was significantly reduced in SC treated with NK-2.1 antiserum and C, whereas SC treated with NK-1.1 antiserum and C showed either less reduction or no reduction in activity against these two cell lines. With two other targets, E male G2 and RBL-5, neither serum produced significant depletion of activity, Analysis of SC indirectly labeled with either NK-1.1 or NK-2.1 antiserum and fluorescein-labeled goat anti-mouse Ig, however, did not detect significant differences between NK-1+ and NK-2+ cell populations.     

Lack of dendritic Thy-1+ epidermal cells in mice with severe combined immunodeficiency disease

C.B-17 scid (severe combined immunodeficiency disease) mice were used to evaluate the relationship of dendritic Thy-1+ epidermal cells (EC) to T lymphocytes (deficient in scid) and to NK cells (replete in scid). Epidermis from scid mice was deficient in dendritic Thy-1+ cells as determined by immunofluorescent staining of epidermal whole mounts. Similarly, epidermal cell suspensions from scid mice failed to proliferate in response to Con A, as compared with epidermal cell suspensions from C.B-17 control mice. Transplantation of normal bone marrow into scid mice reconstituted morphologically identifiable dendritic Thy-1+ EC in whole mounts, as well as Con A responsiveness of EC suspensions, thus indicating that the deficiency in dendritic Thy-1+ EC in scid mice is at the precursor level. These studies demonstrate that Thy-1+ EC are more closely related to T lymphocytes than to NK cells.     

Liver CD4-CD8- NK1.1+ TCR alpha beta intermediate cells increase during experimental malaria infection and are able to exhibit inhibitory activity against the parasite liver stage in vitro

Experimental infection of C57BL/6 mice by Plasmodium yoelii sporozoites induced an increase of CD4-CD8- NK1.1+ TCR alpha beta int cells and a down-regulation of CD4+ NK1.1+ TCR alpha beta int cells in the liver during the acute phase of the infection. These cells showed an activated CD69+, CD122+, CD44high, and CD62Lhigh surface phenotype. Analysis of the expressed TCRV beta segment repertoire revealed that most of the expanded CD4-CD8- (double-negative) T cells presented a skewed TCRV beta repertoire and preferentially used V beta 2 and V beta 7 rather than V beta 8. To get an insight into the function of expanded NK1.1+ T cells, experiments were designed in vitro to study their activity against P. yoelii liver stage development. P. yoelii-primed CD3+ NK1.1+ intrahepatic lymphocytes inhibited parasite growth within the hepatocyte. The antiplasmodial effector function of the parasite-induced NK1.1+ liver T cells was almost totally reversed with an anti-CD3 Ab. Moreover, IFN-gamma was in part involved in this antiparasite activity. These results suggest that up-regulation of CD4-CD8- NK1.1+ alpha beta T cells and down-regulation of CD4+ NK1.1+ TCR alpha beta int cells may contribute to the early immune response induced by the Plasmodium during the prime infection.     

Generation of cytotoxic cells from murine bone marrow by human recombinant IL 2

Murine bone marrow (BM) cells were cultured in recombinant IL 2 (rIL 2) and interferon-alpha, -beta, and -gamma, and cytotoxic activity against YAC cells was determined in a 4-hr 51Cr-release assay. rIL 2 at 20 U/ml was the only lymphokine that consistently induced significant cytotoxic activity within 3 days of culture, peaking around 5 to 7 days. The cytotoxic cells generated are heterogeneous, consisting of at least two populations of cells: a) NK-1+, Qa-5+, AsGm-1+ Thy-1+/-, Lyt-2- cells, similar to natural killer (NK) cells, and b) NK-1-, Qa-5+, AsGm-1+ Thy-1+, Lyt-2+ cells, similar to cytotoxic T lymphocytes. The precursor/accessory cells of these BM cytolytic cells maintained in 20 U/ml of rIL 2 were Qa-2+, Qa-5+, Thy-1+/-, AsGM-1+/-, and NK-1+/- but Lyt-2-. They also lysed NK-resistant targets, P815 and BW5147, and the antigenic phenotypes of these cells were similar to those that lysed YAC cells. These studies indicate that IL 2 alone is adequate to generate cytotoxic activity from BM and that these cytotoxic cells were similar to splenic NK cells.     

Occurrence of mature B (IgM+, B220+) and T (CD3+) lymphocytes in scid mice

Scid mice with and without detectable serum Ig (scid Ig+ and scid Ig- mice, respectively) were examined for the presence of mature "leaky" lymphocytes by flow microfluorimetry with the use of antibodies to B (IgM, B220) and T (CD3, CD4, CD8) lymphocyte surface Ag. The data showed that leaky scid mice are more frequent than is evident from serum Ig analysis and that the incidence of detectable B and T cells increases with age. IgM+ B220+ cells were not detectable in young adult mice (3 mo old), but in old mice (greater than or equal to 1 yr old) they were routinely present in the peritoneal cavity though not in the spleen. Striking differences in the representation of T cell subsets were seen in the thymus of these two age groups. Most young adult mice contained CD3- populations of CD4/CD8 double positive cells, and in some cases, CD4 or CD8 single positive cells as well. By contrast, identifiable T lineage cells in old mice were all CD3+ and predominantly single positive for CD4 or CD8. Detectable peripheral T cell populations numbered less than 10(5) cells, and the representation of T subset markers (CD4, CD8) varied widely among individual mice; further, Southern blot analysis of TCR gene rearrangements in the DNA of polyclonally stimulated lymphoid cultures from these mice showed very restricted heterogeneity relative to that of cultures from normal mice. We conclude that most leaky mice contain very few T cell clones.     

Identification of murine natural killer cell subsets with monoclonal antibodies derived from 129 anti-C57BL/6 immune spleen cells

Two hybridomas producing monoclonal antibodies reactive with natural killer cells were selected after fusion of 129 anti-C57BL/6 immune spleen cells with P3X63-Ag8.653 myeloma cells. Treatment of normal or stimulated cells with the 4LO3311 or the 4LO439 mAb and rabbit complement inhibited natural killer and antibody-dependent cellular cytotoxicities, whereas cell lysis mediated by natural cytotoxic cells, cytotoxic T lymphocytes, or activated macrophages was unaffected. Lymphokine-activated killer activity was reduced after complement-mediated treatment of interleukin-2-stimulated spleen cells with the 4LO3311 mAb but not after treatment with the 4LO439 mAb. Similar treatment of spleen cells with either mAb had no effect on the mitogen-induced proliferation of T and B lymphocytes and did not alter the frequency of antibody plaque-forming cells in immune spleen cell suspensions. The 4LO3311 and 4LO439 mAbs thus appear to be specific for NK cells and their progeny. Flow cytometry analysis confirmed that 4LO3311+ and 4LO439+ cells are phenotypically identical to NK-1.1+ cells. The epitope recognized by the 4LO3311 mAb has the same strain distribution as the NK-2.1 alloantigen previously detected with NZB anti-BALB/c antiserum, whereas the 4LO439 mAb appears to identify a new NK cell marker exclusively expressed in mice of C57BL lineage. The relationship of the molecules detected with either the 4LO3311 or the 4LO439 mAb to polymorphic antigens of the Ly series is discussed.     

Cardiovascular adaptations of pregnancy in T and B cell-deficient mice

The pathophysiology of gestational hypertensive disorders is incompletely defined. T lymphocytes are implicated. Both T and natural killer (NK) cells express RAS and, in implantation sites, NK cells are highly enriched. We hypothesized that T cells and/or NK cells contribute to circulatory control during pregnancy. Using radiotelemetry of arterial pressure, heart rate, and activity, mice without T and B cells (genotypes BALB/c-Rag2(-/-) and NOD.scid) were examined at baseline and across pregnancy. These strains differ in NK cell competency, with Rag2(-/-) being normal and NOD.scid impaired. Circulatory features differed between these inbred strains. Rag2(-/-); had blood pressure responses to pregnancy that did not differ from congenic normal mice. NOD.scid had higher midgestational blood pressure compared with normoglycemic NOD mice (3-5 mm Hg greater than NOD; P < 0.004). In comparison to controls, both T and B strains had much higher heart rates after first trimester that did not remit until parturition (>30 bpm greater than control; P < 0.0001). NOD.scid had additional anomalies, including 90% depletion of circulating NK cells and elevated (57%) proliferation of uterine NK cells within implantation sites. These data demonstrate immune control of midgestational heart rate and suggest NK cells contribute to midpregnancy regulation of mean arterial pressure.     

Immunoregulatory function of CD3+, CD4-, and CD8- T cells. Gamma delta T cell receptor-positive T cells from nude mice abrogate oral tolerance

Previous work has shown that abrogation of oral tolerance is mediated by T cells which are found in the CD3+, L3T4- (CD4-), and Lyt-2- (CD8-) subset (termed double-negative; DN) in mice. Inasmuch as it is known that athymic, nude (nu/nu) mice possess Thy 1+, CD4-, and CD8- T cells which also exhibit a functionally rearranged TCR gamma-chain, we investigated whether this subset of nude T cells contained functional immunoregulatory cells. In this report, we examined the phenotype and distribution of CD3+ T cells in the spleen and in the mesenteric and peripheral lymph nodes of BALB/c nu/nu mice in comparison with normal mice (+/+). In the spleens of nude mice, the predominant CD3+ T cell subpopulation was DN. Further, in mesenteric and peripheral lymph nodes, approximately one-third and one-half of the CD3+ T cells were double negative, respectively. In contrast, CD3+, DN T cells represent a small subpopulation in normal (+/+) mice. We next showed that functional regulatory T cells which possess the ability to abrogate oral tolerance were induced in nu/nu mice by Ag priming. BALB/c nude mice were immunized with SRBC, and the splenic CD3+, Vicia villosa-adherent cells were obtained by panning. Adoptive transfer of CD3+, V. villosa-adherent T cells to orally tolerant BALB/c mice restored responsiveness to SRBC, whereas V. villosa nonadherent cells were without effect. In other experiments, CD3+ T cells from the spleens of SRBC-primed mice were further enriched for the CD5+, DN phenotype and adoptive transfer of this subset completely abrogated oral tolerance to SRBC. To characterize the nature of the TCR expressed on these CD3+, DN T cells, we developed a rabbit antibody to a synthetic peptide (residues 209-218: Tyr-Ala-Asn-Ser-Phe-Asn-Asn-Glu-Lys-Leu) which was synthesized from a deduced sequence of the murine delta-gene. Immunoprecipitation of a cell membrane fraction from CD3+, DN T cells with anti-delta TCR antibody isolated a 45-kDa band. Furthermore, immunoprecipitation of these cells with anti-CD3 (145-2C11) revealed bands at 45 and 35 kDa (corresponding to delta- and gamma-chains, respectively). Taken together, these results are the first to show that gamma delta-TCR bearing CD3+, CD4-, and CD8- T cells are functional and reverse oral tolerance when adoptively transferred.     

The expression of T cell receptor-associated proteins during T cell ontogeny in man

The expression of TCR-associated molecules was examined in human fetal and postnatal tissues. From gestational wk 7 onward in the fetal liver, putative prothymocytes have been identified with cytoplasmic CD3 positivity (cCD3+). These immature cells are TdT- and do not express membrane CD3 (mCD3-) or TCR beta identified by beta F1, but show CD7 and CD45 positivity without CD1, CD2, CD5, CD4, CD8, CD10, and class II Ag. Their high proliferative activity is indicated by greater than 85% Ki67 positivity. After the 10th wk, beta F1+, mCD3+ cells also appear in the liver and these are mostly Ki67- but no TCR gamma delta-bearing cells can be identified at such an early stage of extrathymic development. In the mCD3- TdT-fetal thymus (10 1/2 to 18th wk) cCD3+, mCD3- CD1-blasts proliferate (Ki67+) and lack TCR-beta or TCR-gamma delta. The TdT-, CD1+ cortical thymocytes develop into TCR-beta + and WT31-positive (TCR-alpha beta +) cells. Subsequently TdT-positive thymocytes become detectable around 19 to 20 wk, and in such glands the peak of proliferative activity is seen among TdT+, cCD3+ cells which appear to acquire, in a regular sequence, cytoplasmic beta F1 (TCR-beta), mCD3, and TCR-alpha beta (WT31 positivity) together with the loss of TdT and Ki67 positivity. A newly described transitional population of cells is TdT-, beta F1+ but exhibits no detectable WT31 positivity. These cells correspond to the CD1+, mCD3+ thymocytes and are probably the targets of thymic selection. The cells of the TCR-gamma delta lineage, detected by mAb TCR-delta-1 and delta TCS1, are rare (0.02 to 0.5%) among thymocytes from gestational wk 10 1/2 onward through the whole span of thymic development, but these cells include a proportion (18 to 59%) of cells expressing CD1 Ag, suggesting that these TCR-gamma delta cells differentiate in the thymus. Among the CD1+, TCR-gamma delta + thymocytes, no TdT positivity can be detected.     

Regulation of the secondary in vitro antibody response by endogenous natural killer cells: kinetics, isotype preference, and non-identity with T suppressor cells

Our previous studies had demonstrated that depletion of endogenous natural killer (NK) cells resulted in an augmented primary antibody response in vivo and in vitro. We have now examined the effect of NK cell depletion on the in vitro secondary response to antigen. Treatment of primed murine spleen cells with anti-NK-1.1 allo-antibody and complement before culture resulted in a significant increase in the magnitude of the antigen-specific plaque-forming cell (PFC) response. This treatment did not affect the proportions of Lyt-2+, L3T4+, or sIg+ cells in the population, however, indicating that the augmentation in PFC was not due to changes in the ratio of T to B cells. Removal of endogenous NK cells had a greater effect on the IgG (indirect) PFC response (100 to 200% increase) than on the IgM (direct) PFC response (25 to 50% increase). In contrast, removal of Lyt-2+ cells before culture affected the IgM and IgG responses similarly. Moreover, the kinetics of augmentation differed between cultures depleted of Lyt-2+ cells and those depleted of NK-1.1+ cells. NK cells appeared to act earlier in the response than did T suppressor cells. The NK-1.1+ cells involved in antibody regulation were not involved in the generation of the in vitro derived T suppressor cells. The conclusion that the regulation of the antibody response by NK-1.1+ cells is distinct from that involving T suppressor cells was confirmed in experiments in which removal of both regulatory cell populations resulted in an increase in PFC that was greater than in cultures depleted of either NK or T suppressor cells.     

IL-7 up-regulates IL-4 production by splenic NK1.1+ and NK1.1- MHC class I-like/CD1-dependent CD4+ T cells.

NK T cells are an unusual subset of T lymphocytes. They express NK1. 1 Ag, are CD1 restricted, and highly skewed toward Vbeta8 for their TCR usage. They express the unique potential to produce large amounts of IL-4 and IFN-gamma immediately upon TCR cross-linking. We previously showed in the thymus that the NK T subset requires IL-7 for its functional maturation. In this study, we analyzed whether IL-7 was capable of regulating the production of IL-4 and IFN-gamma by the discrete NK T subset of CD4+ cells in the periphery. Two hours after injection of IL-7 into mice, or after a 4-h exposure to IL-7 in vitro, IL-4 production by CD4+ cells in response to anti-TCR-alphabeta is markedly increased. In contrast, IFN-gamma production remains essentially unchanged. In beta2-microglobulin- and CD1-deficient mice, which lack NK T cells, IL-7 treatment does not reestablish normal levels of IL-4 by CD4+ T cells. Moreover, we observe that in wild-type mice, the memory phenotype (CD62L-CD44+) CD4+ T cells responsible for IL-4 production are not only NK1.1+ cells, but also NK1.1- cells. This NK1.1-IL-4-producing subset shares three important characteristics with NK T cells: 1) Vbeta8 skewing; 2) CD1 restriction as demonstrated by their absence in CD1-deficient mice and relative overexpression in MHC II null mice; 3) sensitivity to IL-7 in terms of IL-4 production. In conclusion, the present study provides evidence that CD4+MHC class I-like-dependent T cell populations include not only NK1.1+ cells, but also NK1.1- cells, and that these two subsets are biased toward IL-4 production by IL-7.