Intracellular pH and the cell cycle of mitogen-stimulated murine lymphocytes

Rapidly proliferating, polyclonally stimulated mouse spleen lymphocytes were separated by density-gradient unit-gravity sedimentation. The following measurements were made on each fraction: the average intracellular water volume, the distribution of DNA content by flow microfluorometry, the rate of ³H-thymidine incorporation, and the intracellular pH. Fractions of cells with a small average intracellular volume were predominately in G0 or G1 phase of the cell cycle, while fractions of larger cells had higher proportions of cells in S or G2. Multiple regression analysis of the data for both T and B lymphocytes indicated that the intracellular pH of cells in G0, G1, or G2 is around pH 7.2, and that the intracellular pH of cells in S phase of the cell cycle is around pH 7.4.     

Loosening of cell cycle controls of human lymphocytes under the action of tumour promoter TPA

The effect of tumour promoter TPA (12-O-tetradecanoylphorbol-13-acetate) on the cell cycle of human peripheral blood lymphocytes stimulated by phytohaemagglutinin (PHA) in vitro was studied and it was found that TPA caused cells to accumulate in all the cell cycle phases. This accumulation took place preferentially at later culture passages, when lymphocytes stimulated by PHA alone stopped mainly in G0/G1 phases. Other effects of TPA were cell induction to enter higher DNA ploidy and to survive and even synthesize DNA under colchicine block of mitosis or under cytochalasin block of cytokinesis. In addition, in experiments in which a transitory block through the G1 phase of cell cycle was applied with use of aminopterin, we could show that a fraction of TPA-treated cells still entered the active phase of DNA synthesis. These findings suggest that TPA causes cell cycle controls to become loose, thereby enhancing adaptability of human lymphocytes to various hindrances in the course of cell cycle and eventually causing them to acquire characteristics known to be common for tumour cells.     

Distinctive expression of the T4 antigen in normal and stimulated lymphocytes

Correlated light scatter and fluorescence flow cytometric analysis of human peripheral blood lymphocytes showed that the expression of the T4 antigen was higher in the larger lymphocytes than in the smaller lymphocytes. A similar expression pattern was observed for HLA Class I antigens but not for T3 and T8, whose expression was independent of cell size. Results with lymphocytes from spleen, lymph node, and tonsil were comparable to those of peripheral blood. Thymocytes, however, were smaller and expressed less T4 and T8 than peripheral lymphocytes. In studies of lymphocytes stimulated in vitro with allogeneic cells or pokeweed mitogen, two populations of T4-positive cells were observed: one of large cells expressing high amounts of T4 and one of small cells expressing low amounts of T4. Similar patterns were seen with T8, although less consistently. In contrast, the expression of T3 was the same in both large and small cells. The large cells expressing high amounts of T4 were not restricted to cells engaged in DNA synthesis or mitosis. This was established by selectively analyzing cells in the G0G1 phases of the cell cycle and by studying stimulated lymphocytes no longer undergoing proliferation. Taken together, these results suggest that immature T lymphocytes are small and express low amounts of T4 and T8. We postulate that as they differentiate, cell size and T4 expression increase proportionally, both parameters increasing even further after antigenic or mitogenic stimulation. The quantitative expression of T4, and probably of T8 but not of T3, is therefore intimately related to maturation and activation of lymphocytes, a fact that may conceivably be related to a functional role of these surface molecules.     

Single-cell analysis of the relationship among transferrin receptors, proliferation, and cell cycle phase in K562 cells

Multiparameter single-cell analysis by flow cytometry was used to distinguish between size-related changes in K562 cell transferrin receptor (TfR) expression and changes in membrane receptor density throughout the cell cycle and over time in culture. Light-scatter pulse-width time-of-flight, a direct and readily calibrated measure of cell diameter, was used to calculate receptor density as the average number of receptors per unit cell surface area. Cell surface TfRs were unimodally distributed over the cell population and were present throughout the cell cycle. The number of receptors increased as cells progressed through the cell cycle, but cell cycle phase was also correlated with cell volume. However, when size heterogeneity was factored out by reanalysis of listmode data, there was a clear cell-cycle effect: among cells of the same size, both the number of receptors per cell and the receptor density increased from G1 to S to G2/M. TfR expression was also followed over time in culture after dilution into fresh medium. A decrease in growth rate after four days was preceded by one to two days by a decrease in both number of TfRs per cell and mean receptor density, indicating that decreased TfR expression represented true "down-regulation" and not just decreased cell size or an increase in the proportion of smaller G1 cells. This type of analysis is generally applicable for resolving the effects of cell size heterogeneity and cell cycle on membrane protein distribution and for other studies of ligand-receptor interaction.     

Different expression profiles of human cyclin B1 in normal PHA-stimulated T lymphocytes and leukemic T cells

BACKGROUND: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes. METHODS: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results. RESULTS: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis. CONCLUSIONS: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.     

Cell cycle analysis of foreign gene (beta-galactosidase) expression in recombinant mouse cells under control of mouse mammary tumor virus promoter

The cell cycle dependency of foreign gene expression in recombinant mouse L cells was investigated. Two different recombinant mouse L cell lines having the glucocorticoid receptor-encoding gene and the lacZ reporter gene were used in this study. The lacZ gene expression was controlled by the glucocorticoid-inducible mouse mammary tumor virus (MMTV) promoter in both cell lines. In "M4" cells the gr gene was under the control of another MMTV promoter, but in "R2" cells it was under the control of the constitutive Rous sarcoma virus promoter. These normally attachment-grown cells were adapted to suspension culture, and a dual-laser flow cytometer was used to simultaneously determine the DNA and foreign protein (beta-galactosidase) content of single living cells. Expression of beta-galactosidase as a function of cell cycle phase was evaluated for cells in exponential growth without any addition of the glucocorticoid inducer, dexamethasone. Cell cycle positions in the S phase were estimated on the basis of DNA content per cell, and position in the G1 phase was estimated on the basis of cell size as measured by pulse-width time of flight. The results showed that beta-galactosidase synthesis occurred through all cell cycle phases, but the expression rate in the G1 phase was much lower than that in the S and G2/M phases in both cell lines. On the basis of cell size analysis, beta-galactosidase expression in M4 cells (with autoinducible promoter) was found to be higher than that in R2 cells (with inducible promoter) during the G1 phase. (c) 1996 John Wiley & Sons, Inc.     

Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase.

Saccharomyces cerevisiae contains 50-100 copies per cell of a circular plasmid called 2 micron DNA. Replication of this DNA was studied in two ways. The distribution of replication events among 2 micron DNA molecules was examined by density transfer experiments with asynchronous cultures. The data show that 2 micron DNA replication is similar to chromosomal DNA replication: essentially all 2 micron duplexes were of hybrid density at one cell doubling after the density transfer, with the majority having one fully dense strand and one fully light strand. The results show that replication of 2 micron DNA occurs by a semiconservative mechanism where each of the plasmid molecules replicates once each cell cycle. 2 micron DNA is the only known example of a multiple-copy, extrachromosomal DNA in which every molecule replicates in each cell cycle. Quantitative analysis of the data indicates that 2 micron DNA replication is limited to a fraction of the cell cycle. The period in the cell cycle when 2 micron DNA replicates was examined directly with synchronous cell cultures. Synchronization was accomplished by sequentially arresting cells in G1 phase using the yeast pheromone alpha-factor and incubating at the restrictive temperature for a cell cycle (cdc 7) mutant. Replication was monitored by adding 3H-uracil to cells previously labeled with 14C-uracil, and determining the 3H/14C ratio for purified DNA species. 2 micron DNA replication did not occur during the G1 arrest periods. However, the population of 2 micron DNA doubled during the synchronous S phase at the permissive temperature, with most of the replication occurring in the first third of S phase. Our results indicate that a mechanism exists which insures that the origin of replication of each 2 micron DNA molecule is activated each S phase. As with chromosomal DNA, further activation is prevented until the next cell cycle. We propose that the mechanism which controls the replication initiation of each 2 micron DNA molecule is identical to that which controls the initiation of chromosomal DNA.     

BrdU-Hoechst flow cytometry reveals regulation of human lymphocyte growth by donor-age-related growth fraction and transition rate

An improved BrdU-Hoechst flow assay was applied to cell kinetic studies of human lymphocyte cultures during a 24-96 hr interval after PHA stimulation. The assay shows that the duration of the initial lag phase and the proportions of noncycling cells increase as a function of donor age, whereas the rates of transition from each cell cycle compartment to the next decrease. Cell cycle arrest occurs in the first S and G2 phase after stimulation of lymphocytes from a 75-year-old donor but not from younger donors. The data are consistent with several models of cell cycle kinetics, so long as these models are modified to include a fraction of noncycling cells in each cell cycle compartment.     

A quantitative evaluation of the changes in the proliferation of a human lymphocyte culture after mutagen exposure at the G0 stage

Cell cycle delay of human lymphocytes treated with mutagens at G0 stage has been studied using a mathematical model. Cell cycle delay depends on entry cells into the first S phase and is independent on the cell cycle duration.     

Differences in centromere positioning of cycling and postmitotic human cell types

Centromere positioning in human cell nuclei was traced in non-cycling peripheral blood lymphocytes (G0) and in terminally differentiated monocytes, as well as in cycling phytohemagglutinin-stimulated lymphocytes, diploid lymphoblastoid cells, normal fibroblasts, and neuroblastoma SH-EP cells using immunostaining of kinetochores, confocal microscopy and three-dimensional image analysis. Cell cycle stages were identified for each individual cell by a combination of replication labeling with 5-bromo-2-deoxyuridine and immunostaining of pKi67. We demonstrate that the behavior of centromeres is similar in all cell types studied: a large fraction of centromeres are in the nuclear interior during early G1; in late G1 and early S phase, centromeres shift to the nuclear periphery and fuse in clusters. Peripheral location and clustering of centromeres are most pronounced in non-cycling cells (G0) and terminally differentiated monocytes. In late S and G2, centromeres partially decluster and migrate towards the nuclear interior. In the rather flat nuclei of adherently growing fibroblasts and neuroblastoma cells, kinetochores showed asymmetrical distributions with preferential kinetochore location close either to the bottom side of the nucleus (adjacent to the growth surface) or to the nuclear upper side. This asymmetrical distribution of centromeres is considered to be a consequence of chromosome arrangement in anaphase rosettes.     

Desferoxamine blocks IL 2 receptor expression on human T lymphocytes

Thymidine uptake by PHA-stimulated human lymphocytes is reduced in the presence of 100 microM or greater concentrations of the iron-chelating agent desferoxamine (DF). We assessed expression of IL 2 receptor, 4F2 and Ia antigens, IL 2 production, and cell cycle progression by blood mononuclear cells (MNC) stimulated by PHA in the presence or absence of DF to determine whether the lack of T cell proliferation was a manifestation of inhibition of an earlier activation event. Tac antigen expression on PHA-stimulated MNC was inhibited by DF throughout 8 days of culture, and those cells which were positive had a low density of Tac antigen as compared with controls without DF. Expression of other activation antigens, 4F2 and Ia, was not impaired by DF. The supernatants of the DF-containing and control cultures contained equivalent IL 2 activity, as measured on the HT-2 cell line. Cell cycle analysis of these cultures shows that the addition of DF at the beginning of culture blocks most cells from undergoing G0 to G1 transition, whereas later addition of DF arrests the progression of the T cell blasts through the cell cycle. Separation of cells cultured with PHA and DF into Tac+ and Tac- subsets showed that progression from G0 to G1 was restricted to the former subset. These results suggest that interference with IL 2 receptor expression might contribute to the block in mitogen-induced proliferation caused by DF.     

Size distribution and hormonal responsiveness of dispersed rabbit luteal cells during pseudopregnancy

Due to the evidence for two distinct steroidogenic cell types in corpora lutea of large domestic animals, cells of the rabbit corpus luteum were characterized with respect to cell diameters, relative abundance, steroidogenic capacity and responsiveness to hormones. Pseudopregnancy was induced in New Zealand rabbits by injection of 30-160 IU pregnant mare's serum gonadotropin (PMSG) followed in 2-4 days by an i.m. injection of 20-35 micrograms gonadotropin-releasing hormone (GnRH). Corpora lutea were obtained 2, 5 and 9 days after injection of GnRH and dissociated into single cell suspensions. Suspended steroidogenic cells were incubated (2 h, 37 degrees C) in medium 199 alone or in medium containing ovine luteinizing hormone (oLH) (100 ng/ml), or isoproterenol (100 microM). Media were collected and assayed for progesterone content. Secretion of progesterone (means +/- SE, n = 4) was stimulated (p less than 0.05) by oLH on each day: Day 2 = 1.7 +/- 0.2-fold; Day 5 = 3.5 +/- 0.4-fold; and Day 9 = 3.1 +/- 0.6-fold stimulation above controls. Isoproterenol also stimulated (p less than 0.05) secretion of progesterone by suspended luteal cells on Days 2 and 9. Microscopic examination of cell suspensions stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity provided identification of cells with steroidogenic capacity. The diameters (means +/- SE) for steroidogenic cells increased (p less than 0.05) from Days 2 to 9 (Day 2 = 15.2 +/- 0.2 micron; Day 5 = 22.4 +/- 0.4 micron; Day 9 = 28.3 +/- 1.6 micron). The large cell to small cell ratio increased from 0.01 on Day 2 to 2.03 on Day 9.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytofluorometric determination of protein-bound thiols and DNA in cell nuclei

The aim of the present study was to establish a cytofluorometric method for the simultaneous determination of protein-bound sulfhydryl-groups (PSH) and DNA in isolated cell nuclei. DNA was stained with ethidiumbromide and PSH with N-iodoacetyl-N(5-sulfo-1-naphthyl) ethylendiamine (AEDANS). Disulfide groups of nuclear proteins were determined by the same method after reduction with sodium borohydride or thioglycollic acid. The method was established by using nuclei of human lymphocytes, which then served as a biological standard for further investigations of the nuclei of different mammalian cell types: nuclei from mouse liver cells and nuclei from the cells of two human melanoma cell lines. For non-proliferating lymphocytes distinct DNA- and PSH-values could be measured. The PSH-values detected in the nuclei of the other cell types were higher by comparison and varied within the cell cycle; i.e., PSH increased during the S-phase and was almost doubled during the cell generation cycle from G1- to G2-phase. Cell line and cell cycle-dependent variations of nuclear disulfides could also be detected. These results are discussed with respect to their radiobiological implications. In conclusion, thiol groups may represent one factor determining the radiosensitivity of cells, but they are not the only decisive one.     

Macrophage-mediated cytostatic activity blocks lymphoblast cell cycle progression independently in both G1 phase and S phase

Recent work has shown that macrophage-mediated cytostatic activity inhibits cell cycle traverse in G1 and/or S phase of the cell cycle without affecting late S, G2, or M phases. The present report is directed at distinguishing between such cytostatic effects on G1 phase or S phase using the accumulation of DNA polymerase alpha as a marker of G1 to S phase transition. Quiescent lymphocytes stimulated with concanavalin A undergo a semisynchronous progression from G0 to G1 to S phase with a dramatic increase in DNA polymerase alpha activity between 20 and 30 hr after stimulation. This increase in enzyme activity was inhibited, as was the accumulation of DNA, when such cells were cocultured with activated murine peritoneal macrophages during this time interval. However, if mitogen-stimulated lymphocytes were enriched for S-phase cells by centrifugal elutriation and cocultured with activated macrophages for 4-6 hr, DNA synthesis was inhibited but the already elevated DNA-polymerase activity was unaffected. Similar results were obtained when a virally transformed lymphoma cell line was substituted as the target cell in this assay. These results show that both G1 and S phase of the cycle are inhibited and suggest that inhibition of progression through the different phases may be accomplished by at least two distinct mechanisms.     

Increased voltage-gated potassium conductance during interleukin 2- stimulated proliferation of a mouse helper T lymphocyte clone

Recent work has demonstrated the presence of voltage-gated potassium channels in human peripheral blood T lymphocytes (Matteson, R., and C. Deutsch, 1984, Nature (Lond.), 307:468-471; DeCoursey T. E., T. G. Chandy, S. Gupta, and M. D. Cahalan, 1984, Nature (Lond.), 307:465-468) and a murine cytolytic T-cell clone (Fukushima, Y., S. Hagiwara, and M. Henkart, 1984, J. Physiol., 351:645-656). Using the whole cell patch clamp, we have found a potassium conductance with similar properties in a murine noncytolytic T lymphocyte clone, L2. Under voltage clamp, a step from a holding potential of -70 mV to +50 mV produces an average outward current of 100-150 pA in "quiescent" L2 cells at the end of their weekly maintenance cycle. When these cells are stimulated with human recombinant interleukin 2 (rIL2, 100 U/ml), they grow in size and initiate DNA synthesis at approximately 24 h. Potassium conductance is increased as early as 8 h after stimulation with rIL2 and rises to a level 3-4 times that of excipient controls by 24 h. The level remains elevated through 72 h, but as the cells begin to leave the cell cycle at 72-96 h, the conductance decreases quickly to a value only slightly higher than the initial one. Quinine, a blocker of this conductance, markedly reduces the rate at which L2 cells traverse the cell cycle, while also reducing the rate of stimulated protein synthesis. The regulation of potassium conductance in L2 cells during rIL2-stimulated proliferation suggests that potassium channel function may play a role in support of the proliferative response.     

EGF-dependent epithelial cells of the mammary cell line HC11 demonstrate a high degree of synchronized cell cycle during stimulation with epidermal growth factor

The most popular object for studying endocytosis of EGF-receptor complexes, human epidermoid carcinoma A431, was shown to answer to EGF in high concentration (100 ng/ml) by growth inhibition, being indifferent to lower (0.1-1 ng/ml) concentrations. At the same time, cells NIH 3T3, expressing human EGF receptor (HER14), and epithelial mammary cells HC11 increased 14C-thymidine incorporation into DNA after EGF addition. However, for HER14 cells stimulatory effect of EGF was twice weaker than that induced by serum, whereas the effect of EGF on 14C-thymidine incorporation in DNA of cells HC11 was approximately 5 times stronger compared to serum. Therefore, cells HC11 may be referred to as EGF-dependent. Cell cycle analysis by fluorimetry showed that more than 90% of serum-starved HER14 and HC11 were in G0/G1. Within 19-20 h after stimulation by EGF 70-90% of HC11 cells and only 30-40% of HER14 cells were in S-phase. EGF removing from culture medium earlier than 9-11 h after stimulation blocked entering of HC11 cells into S-phase, whereas such EGF-dependent period was not found for cells HER14. Thus, synchronization of progression through early stages of cell cycle, stimulated by EGF and the presence of well defined "early" (EGF-dependent) and "late" (EGF-independent) phases, make cells HC11 convenient object for studying physiological role of EGF receptor complexes endocytosis.     

Elevation of Survivin Levels by Hematopoietic Growth Factors Occurs in Quiescent CD34+ Hematopoietic Stem and Progenitor Cells Before Cell Cycle Entry

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is over-expressed during G2/M phase in most cancer cells. In contrast, we previously reported that Survivin is expressed throughout the cell cycle in normal CD34+ hematopoietic stem and progenitor cells stimulated by the combination of Thrombopoietin (Tpo), Stem Cell Factor (SCF) and Flt3 ligand (FL). In order to address whether Survivin expression is specifically up-regulated by hematopoietic growth factors before cell cycle entry, we isolated quiescent CD34+ cells and investigated Survivin expression in response to growth factor stimulation. Survivin is up-regulated in CD34+ cells with 2N DNA content following growth factor addition, suggesting it becomes elevated during G0/G1. Survivin is barely detectable in freshly isolated umbilical cord blood (UCB) Ki-67negative and Cyclin Dnegative CD34+ cells, however incubation with Tpo, SCF and FL for 20 hrs results in up-regulation without entry of cells into cell cycle. Culture of G0 CD34+ cells isolated based on Hoechst 33342/PyroninY staining with Tpo, SCF and FL for 48 hrs, results in significantly elevated Survivin mRNA and protein levels. Moreover, labeling of fresh G0 CD34+ cells with 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) before culture with growth factors for up to 72 hrs, revealed that Survivin expression was elevated in CFSEbright G0 CD34+ cells, indicating that up-regulation occurred before entry into G1. These results suggest that up-regulation of Survivin expression in CD34+ cells is an early event in cell cycle entry that is regulated by hematopoietic growth factors and does not simply reflect cell cycle progression and cell division.

Key Words:

Survivin, Cord blood, CD34+ cells, Cell cycle     

Cell cycle-dependent AgNOR analysis in invasive breast cancer

OBJECTIVE: To investigate to what extent analysis of silver-stained nucleolar organizer regions (AgNORs) is cell cycle dependent in breast cancer and to assess the prognostic value of an AgNOR analysis that takes into consideration the cell cycle status of tumor cells. STUDY DESIGN: In 97 cases of invasive breast carcinoma, morphometric AgNOR analysis was performed in tumor cells with immunohistochemical MIB-1 reactivity (NORcyc analysis) and in MIB-1-negative tumor cells (NORnon analysis). Additionally, conventional (NORconv) analysis without preceding MIB-1 staining was done. Findings were compared with the Nottingham prognostic index (NPI). RESULTS: In comparison to noncycling tumor cells, cycling ones exhibited significantly higher AgNOR numbers (mean values, 3.84 +/- 1.09 vs. 2.40 +/- 0.78 per nucleus), higher total AgNOR areas (5.95 +/- 3.17 vs. 5.62 +/- 3.05 micron 2, NS) and significantly lower mean AgNOR areas (2.08 +/- 1.14 vs. 2.93 +/- 1.69 micron 2). When related to NPI, correlation coefficients of NORnon analysis were higher than those of NORcyc analysis but lower than those of NORconv analysis. Among the different AgNOR parameters, total AgNOR area correlated best with NPI. CONCLUSION: Cell cycle status has a high impact on AgNOR analysis. However, the best prognostic information in breast cancer is derived from an AgNOR analysis that considers both cycling and noncycling tumor cells.     

Increased nonobese diabetic Th1:Th2 (IFN-gamma:IL-4) ratio is CD4+ T cell intrinsic and independent of APC genetic background

Autoreactive CD4(+) T cells play a major role in the pathogenesis of autoimmune diabetes in nonobese diabetic (NOD) mice. We recently showed that the non-MHC genetic background controlled enhanced entry into the IFN-gamma pathway by NOD vs B6.G7 T cells. In this study, we demonstrate that increased IFN-gamma, decreased IL-4, and decreased IL-10 production in NOD T cells is CD4 T cell intrinsic. NOD CD4(+) T cells purified and stimulated with anti-CD3/anti-CD28 Abs generated greater IFN-gamma, less IL-4, and less IL-10 than B6.G7 CD4(+) T cells. The same results were obtained in purified NOD.H2(b) vs B6 CD4(+) T cells, demonstrating that the non-MHC NOD genetic background controlled the cytokine phenotype. Moreover, the increased IFN-gamma:IL-4 cytokine ratio was independent of the genetic background of APCs, since NOD CD4(+) T cells generated increased IFN-gamma and decreased IL-4 compared with B6.G7 CD4(+) T cells, regardless of whether they were stimulated with NOD or B6.G7 APCs. Cell cycle analysis showed that the cytokine differences were not due to cycle/proliferative differences between NOD and B6.G7, since stimulated CD4(+) T cells from both strains showed quantitatively identical entry into subsequent cell divisions (shown by CFSE staining), although NOD cells showed greater numbers of IFN-gamma-positive cells with each subsequent cell division. Moreover, 7-aminoactinomycin D and 5-bromo-2'-deoxyuridine analysis showed indistinguishable entry into G(0)/G(1), S, and G(2)/M phases of the cell cycle for both NOD and B6.G7 CD4(+) cells, with both strains generating IFN-gamma predominantly in the S phase. Therefore, the NOD cytokine effector phenotype is CD4(+) T cell intrinsic, genetically controlled, and independent of cell cycle machinery.