X-chromosome activity in heterokaryons and hybrids between mouse fibroblasts and teratocarcinoma stem cells

To determine whether 2X-active cells contain factors capable of reactivating the inactive mammalian X chromosome, fibroblast lines, having a cytologically or genetically marked inactive X, were fused with 2X-active mouse embryos or ovarian teratocarcinoma stem cells. Fusions with 2–16 cell embryos were uninformative because no mitosis occurred in heterokaryons. Fusions with 2X-active teratocarcinoma cells, and screening for re-expression of alleles on the inactive X showed that reactivation did not occur with detectable frequency in heterokaryon. Hybridization of HPRT^?M. musculus × M. caroli cells with XO HPRT^? teratocarcinoma cells yielded hybrids with a frequency of >10^?6; these hybrids all expressed the Hpt allele on the inactive M. caroli X, but not the M. caroliGpd or Pgk. Late replication-banding studies of hybrids and 6-thioguanine-resistant revertants showed that the reactivated Hp⁺ allele was still located on the late replicating X. Similar results were obtained with hybridization of this line to 1X-active (male-derived) fibroblast lines, indicating that hybridization per se, rather than a specific factor contributed by the teratocarcinoma cell partner, was reponsible for the frequent localized derepression of the Hpt⁺ allele on the inactive X.     

Expression of SV40 Large T Antigen Stimulates Reversion of a Chromosomal Gene Duplication in Human Cells

Transformation of human cells is characterized by altered cell morphology, frequent karyotypic abnormalities, reduced dependence on growth factors and substrate, and rare “immortalization”—clonal acquisition of unlimited proliferative potential. We previously reported a marked increase in DNA rearrangements, arising between two duplicated segments in a transfected plasmid substrate, for five immortal human cell lines relative to three normal fibroblast strains [Finnet al.(1989)Mol. Cell. Biol.9, 4009–4017]. We have now assessed reversion of a 14-kilobase-pair duplication within the hypoxanthine phosphoribosyl transferase (HPRT) gene locus, in a fibroblast strain during its normal replicative lifespan and after stable transformation with SV40 large-T antigen. Revertants, selected under HPRT-dependent growth conditions immediately after purging preexistingHPRT⁺cells, were confirmed asHPRT⁺by hypoxanthine incorporation and 6-thioguanine sensitivity. Southern blot analyses indicate loss from most revertant clones of a restriction fragment representing the duplicatedHPRTregion, as predicted for homologous recombination between the 14-kilobase-pair repeats. Amplification of a subregion ofHPRTmRNA implicated deletion of duplicated exons in 93% of revertant colonies. Reversion toHPRT⁺was unaltered during the normalin vitrolifespan of these cells, but increased in 9 clones stably transformed with large-T antigen (mean = 3.8-fold; eachP< 10⁻⁵). Stimulation ofHPRT-reversion is abrogated in a variety of T-antigen mutants, and depends on continued induction of T antigen by glucocorticoid in two clones tested 10–30 doublings before replicative senescence. Since no immortal subclones arose from these clones, elevated reversion must precede immortalization. Increased DNA rearrangements, in cells expressing T-antigen, could facilitate the rare concurrence of multiple mutations necessary for immortalization.     

Evidence for a relationship between DNA methylation and DNA replication from studies of the 5-azacytidine-reactivated allocyclic X chromosome

We examined the sequence of DNA synthesis of the human active, inactive and reactivated X chromosomes in mouse-human hybrid cells. The two independent reactivants, induced by 5-azacytidine (5-azaC), expressed human hypoxanthinephosphoribosyl transferase (HPRT), and one also expressed human glucose-6-phosphate dehydrogenase (G6PD) and phosphoglycerate kinase (PGK). Restriction enzyme analysis of DNA methylation at the re-expressed loci revealed hypomethylation of CpG clusters, that characterizes the relevant genes on the active X. The transfer of active and inactive X chromosomes from the native environment of the human fibroblast to the foreign environment of the hybrid cell did not affect the specific replication sequence of either human X chromosome. The silent X chromosome when reactivated, remained allocyclic, and the first bands to replicate were the same as prior to reactivation. In one reactivant, however, further progression of replication was significantly altered with respect to the order in which bands were synthesized. This alteration in the replication of the silent X following 5-azaC-induced reactivation suggests that DNA methylation may modulate the replication kinetics of chromosomal DNA.     

Gene amplification in a mouse embryo? Double minutes in cell lines independently derived from a Mus musculus x M. caroli fetus

Double minutes (DM) were found to be present in six of seven clones derived from a 16-day female Mus musculus x M. caroli fetus. The DM-positive clones derived from three primary populations independently set up from the fetus, and included clones with an active M. caroli X chromosome as well as clones with an active M. musculus X. The simplest explanation of these findings is that DM were already present in cells of the M. musculus x M. caroli embryo at the time of X chromosome inactivation and persisted during in vivo development and in vitro culture. This suggests that gene amplification occurred in the early embryo, or even the fertilized egg, perhaps because of interactions between components of germ cells contributed by the M. musculus and M. caroli parents. Alternatively, induction may have occurred independently in these lines, requiring that amplification is an unusually common occurrence in cells from interspecific hybrids.     

Cotransfer and phenotypic stabilisation of syntenic and asyntenic mink genes into mouse cells by chromosome-mediated gene transfer

Summary By means of metaphase chromosomes, the genes for mink thymidine kinase (TK) and hypoxanthine-phosphoribosyltransferase (HPRT) were transferred to mutant mouse cells, LMTK^-, A9 (HPRT^-) and teratocarcinoma cells, PCC4-aza 1 (HPRT^-). Eighteen colonies were isolated from LMTK^- (series A), 9 from A9 (series B) and none from PCC4-aza 1. The transformed clones contained mink TK or HPRT. Analysis of syntenic markers in series B demonstrated that one clone contained mink glucose-6-phosphate dehydrogenase (G6PD) and the other alpha-galactosidase; in series A, nine clones contained mink galactokinase (GALK) and six mink aldolase C (ALDC). Analysis of 12 asyntenic markers located in ten mink chromosomes showed the presence of only aconitase-1 (ACON1) (the marker of mink chromosome 12) in three clones of series A. The clones lost mink ACON1 between the fifth to tenth passages. Cytogenetic analysis established the presence of a fragment of mink chromosome 8 in eight clones of series A, but not in series B. The clones of series A lost mink TK together with mink GALK and ALDC during back-selection; in B, back-selection retained mink G6PD. No stable TK⁺ phenotype was detected in clones with a visible fragment of mink chromosome 8. Stability analysis demonstrated that about half of the clones of series B have stable HPRT⁺ phenotype whereas only three clones of series A have stable TK⁺ phenotype. It is suggested that the recipient cells, LMTK^- and A9, differ in their competence for genetic transformation and integration of foreign genes.     

Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells

We describe an assay for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10⁶ cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10 μM) is then added directly to the growing culture and AS-resistant (AS^r) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT^? clones. The AS^r phenotype is characterized both physiologically and biochemically. All AS^r clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT. The data provide further evidence that ICR-191 and ICR-170 cause structural gene mutations in mammalian cells and also suggest that ICR-191, ICR-170, and N-ethyl-N-nitrosourea induce similar types of mutations in Chinese hamster ovary cells.     

Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing

Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.     

Chromosome composition of interspecies hybrid embryonic stem cells in mice

Chromosome complements of 20 hybrid clones obtained by fusing Mus musculus embryonic stem cells (ESCs) and Mus caroli splenocytes were studied. The use of two-color fluorescence hybridization in situ with chromosome- and species-specific probes has allowed us to reliably reveal the parental origin of homologs of any chromosome in hybrid cells. Depending on the ratio of parental chromosome homologs, all 20 hybrid clones were separated in several groups ranging from the clones that contain cells that are nearly tetraploid with two diploid sets of M. musculus and single M. caroli chromosomes to clones with a marked predominance of the M. caroli chromosome. In eight hybrid cell clones, we observed the pronounced prevalence of chromosomes of the pluripotent partner over chromosomes of the somatic partner in a ratio of 5: 1 to 3: 1. In other hybrid cell clones, the ratio of M. musculus to M. caroli chromosomes was either equal (1: 1; 2: 2) or with the prevalence of the pluripotent (2: 1) or differentiated (1: 2) partner. In three hybrid cell clones, for the first time, we observed the predominant segregation of ESC-derived pluripotent chromosomes. This might indicate the compensation for the epigenetic differences between parental chromosomes of the ESC and splenocyte origin.     

A mammalian cellular system for the concomitant study of neoplastic transformation and somatic mutation.

A mammalian cellular system, utilizing Syrian hamster embryo cells, was developed for the concomitant study of neoplastic transformation and somatic mutation. Chemically induced somatic mutation of the cells was assayed at two genetic loci. Mutants deficient in hypoxanthine phosphoribosyl transferase (HPRT) were detected by the production of colonies resistant to 8-azaguanine (AG^r) or 6-thioguanine (TG^r) and mutants with an altered Na⁺/K⁺ ATPase were detected by the production of colonies resistant to ouabain (Oua^r). Colonies resistant to each of the three selective agents were isolated and characterized. AG^r and TG^r resistant cells maintained their resistance to the selective agent after isolation and growth in the absence of the drug, displayed a low reversion frequency, and possessed less than 1% of the HPRT activity of the wild-type cells. AG^r cells were also resistant to the cytotoxicity effects of 6TG. Oua^r cells also maintained their resistance to ouabain and were less sensitive to the inhibition of ⁸⁶Rb uptake by ouabain than the wild-type cells. The spontaneous frequency of all three types of resistant cells was <10^?6, but the mutation frequency was significantly increased following exposure of the cells to known mutagens in a dosage-dependent manner. These properties indicate that AG^r and TG^r cells posess a mutation in the structural or regulatory gene for HPRT, and that Oua^r cells have an altered Na⁺/K⁺ ATPase.The factors involved in the quantification of the mutation frequencies of hamster embryo cells following exposure to carcinogens were determined. Cytotoxicity was assayed by a reduction in the cloning efficiency of the treated cells. The recovery efficiencies of the resistant cells were measured by reconstitution experiments and the degree of cross feeding effects of HPRT^? cells was determined. The expression time of the mutations following exposure of the cells to carcinogens was also examined, and the mutation frequencie at the two loci of hamster embryo cells following exposure to MNNG or benzo(a)-pyrene (B(a)P) were determined. Employing this system, a quantitative comparison can be made between the frequencies of somatic mutation and morphological transformation.     

Non-selective isolation of fibroblast-cybrids by flow sorting

Red- and green-fluorescing polystyrene beads were used to label different populations of cultured human fibroblasts. After enucleation of the green-fluorescing population, the cytoplasts were fused with red-fluorescing cells. Twenty-four hours after cell fusion the population of red-green heterofluorescent cells was isolated with a FACS II cell sorter. When Lesch-Nyhan fibroblasts (HPRT⁻) were fused with cytoplasts from control fibroblasts (HPRT⁺) more than 95% of the sorted cells were heterofluorescent and 90% of the sorted cells showed HPRT⁺ activity. Therefore almost all sorted heterofluorescent cells are true cybrids. With this procedure for cybrid isolation, earlier complementation studies using cybrids from different variants of β-galactosidase deficiency could be confirmed.     

Phylogenetic relationship and time of divergence of <Emphasis Type="Italic">Mus terricolor</Emphasis> with reference to other <Emphasis Type="Italic">Mus</Emphasis> species

Mitochondrial DNA control region of Mus terricolor, three aboriginal species M. spretus, M. macedonicus, M. spicilegus; the Asian lineage M. caroli, M. cervicolor, M. cookii; and the two house mice, M. musculus domesticus and M. m. castaneus were analysed to estimate the substitution rate, phylogenetic relationship and the probable time of divergence. Results showed that M. spretus, M. caroli and M. terricolor are highly diverged from each other (caroli/terricolor = 0.146, caroli/spretus = 0.147 and terricolor/spretus = 0.122), whereas M. spretus showed less divergence with two house mice species (0.070 and 0.071). Sequence divergence between M. terricolor and the Palearctic group were found to be ranging from 0.121 to 0.134. Phylogenetic analysis by minimum evolution, neighbour-joining, unweighed pair group method with arithmetic mean and maximum parsimony showed almost similar topology. Two major clusters were found, one included the Asian lineage, M. caroli, M. cookii and M. cervicolor and the other included the house mice M. m. domesticus, M. m. castaneus and the aboriginal mice M. macedonicus and M. spicilegus along with M. spretus, forming the Palearctic clade. M. terricolor was positioned between the Palearctic and Asian clades. Results showed that Palearctic-terricolor and the Asian lineages diverged 5.47 million years ago (Mya), while M. terricolor had split around 4.63 Mya from their ancestor. M. cervicolor, M. cookii and M. caroli diverged between 4.70 and 3.36 Mya, which indicates that M. terricolor and the Asian lineages evolved simultaneously. M. spretus is expected to have diverged nearly 2.9 Mya from their most recent common ancestor.     

Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220+ Double-Negative T Lymphocytes of Autoimmune MRL/lpr Mice

Lupus is a chronic inflammatory autoimmune disease influenced by multiple genetic loci including Fas Ligand (FasL) and P2X7 receptor (P2X7R). The Fas/Fas Ligand apoptotic pathway is critical for immune homeostasis and peripheral tolerance. Normal effector T lymphocytes up-regulate the transmembrane tyrosine phosphatase B220 before undergoing apoptosis. Fas-deficient MRL/lpr mice (lpr mutation) exhibit lupus and lymphoproliferative syndromes due to the massive accumulation of B220⁺ CD4^–CD8^– (DN) T lymphocytes. The precise ontogeny of B220⁺ DN T cells is unknown. B220⁺ DN T lymphocytes could be derived from effector CD4⁺ and CD8⁺ T lymphocytes, which have not undergone activation-induced cell death due to inactivation of Fas, or from a special cell lineage. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of proinflammatory cytokines and TNFR1/Fas-independent cell death. P2X7R also regulate early signaling events involved in T-cell activation. We show herein that MRL/lpr mice carry a P2X7R allele, which confers a high sensitivity to ATP. However, during aging, the MRL/lpr T-cell population exhibits a drastically reduced sensitivity to ATP- or NAD-mediated stimulation of P2X7R, which parallels the increase in B220⁺ DN T-cell numbers in lymphoid organs. Importantly, we found that this B220⁺ DN T-cell subpopulation has a defect in P2X7R-mediated responses. The few B220⁺ T cells observed in normal MRL^+/+ and C57BL/6 mice are also resistant to ATP or NAD treatment. Unexpectedly, while P2X7R mRNA and proteins are present inside of B220⁺ T cells, P2X7R are undetectable on the plasma membrane of these T cells. Our results prompt the conclusion that cell surface expression of B220 strongly correlates with the negative regulation of the P2X7R pathway in T cells.     

Interspecific variation in extracellular polysaccharide content and colony formation of <Emphasis Type="Italic">Microcystis</Emphasis> spp. cultured under different light intensities and temperatures

Single cells of five different Microcystis species (M. ichthyoblabe, M. viridis, M. flos-aquae, M. wesenbergii, and M. aeruginosa) were batch-cultured at different temperatures and light intensities: (a) 25 °C and 50 μmol photons m^?2 s^?1 (control culture); (b) 25 °C and 10 μmol photons m^?2 s^?1; and (c) 15 °C and 50 μmol photons m^?2 s^?1. The extracellular polysaccharide content was significantly higher in treatments b and c than in the control treatment. All Microcystis species existed as single cells under the control treatment but formed colonies in treatments b and c. All of the colonies were irregular with indistinct margins. M. ichthyoblabe, M. viridis, M. flos-aquae, and M. wesenbergii formed colonies with similar morphologies and their cells were loosely aggregated. In contrast, M. aeruginosa formed denser colonies with no distinct holes. The colony morphologies differed from the classic morphology of M. ichthyoblabe field-grown colonies but resembled that of small colonies found in Lake Taihu (Yangtze Delta Plain, China) during early spring. This indicates that field- and laboratory-grown colonies are governed by similar formation processes. We suggest that in laboratory and field environments, M. ichthyoblabe (or M. flos-aquae) colonies are representative of small colonies formed from single Microcystis cells, whereas the morphology of older colonies evolves to resemble M. wesenbergii and M. aeruginosa colonies.     

Plasmid Copy Number Underlies Adaptive Mutability in Bacteria

The origin of mutations under selection has been intensively studied using the Cairns-Foster system, in which cells of an Escherichia coli lac mutant are plated on lactose and give rise to 100 Lac⁺ revertants over several days. These revertants have been attributed variously to stress-induced mutagenesis of nongrowing cells or to selective improvement of preexisting weakly Lac⁺ cells with no mutagenesis. Most revertant colonies (90%) contain stably Lac⁺ cells, while others (10%) contain cells with an unstable amplification of the leaky mutant lac allele. Evidence is presented that both stable and unstable Lac⁺ revertant colonies are initiated by preexisting cells with multiple copies of the F′lac plasmid, which carries the mutant lac allele. The tetracycline analog anhydrotetracycline (AnTc) inhibits growth of cells with multiple copies of the tetA gene. Populations with tetA on their F′lac plasmid include rare cells with an elevated plasmid copy number and multiple copies of both the tetA and lac genes. Pregrowth of such populations with AnTc reduces the number of cells with multiple F′lac copies and consequently the number of Lac⁺ colonies appearing under selection. Revertant yield is restored rapidly by a few generations of growth without AnTc. We suggest that preexisting cells with multiple F′lac copies divide very little under selection but have enough energy to replicate their F′lac plasmids repeatedly until reversion initiates a stable Lac⁺ colony. Preexisting cells whose high-copy plasmid includes an internal lac duplication grow under selection and produce an unstable Lac⁺ colony. In this model, all revertant colonies are initiated by preexisting cells and cannot be stress induced.     

Related satellite DNA's in the genus Mus

Several Thailand species from the genus Mus have been shown to contain satellite DNA's able to cross-reassociate with the Mus musculus satellite. One species, Mus caroli, contains at least three discrete but related light satellite DNA's. All the related Mus satellites band on the light side of the major band in neutral CsCl gradients, separate into complementary strands in alkaline CsCl gradients, and have a relatively low affinity for Ag⁺. Three of the Mus satellite DNA's have been purified: taken separately, they show very sharp thermal transitions and reassociate at similar rates to give well-matched duplexes.     

Transfer of Sensitivity to Tumor Promoters by Transfection of DNA from Sensitive into Insensitive Mouse JB6 Epidermal Cells

Sensitivity to promotion of transformation by tumor promoters in mouse epidermal JB6 cells appears to have a genetic basis since the phenotypes of both promotable and nonpromotable JB6 cells derived from a common parent line are stable. Hybridization of promotable (P⁺) and nonpromotable (P⁻) cells previously indicated that promotability appears to behave as a dominant trait. These results suggest that it should be possible to find DNA sequences which specify sensitivity to promotion of anchorage independence by 12-o-tetradecanoyl-phorbol-13-acetate (TPA). Cellular DNA isolated from one of two P⁺ lines, JB6 Cl 41 or JB6 Cl 22, was CaPO₄ precipitated and used to transfect the P⁻ cell line JB6 Cl 30. At 7 days posttransfection, the cells were suspended in agar with or without TPA at 1.6 × 10⁻⁸ M and assayed 10 days later for TPA-dependent colony formation. Untreated or Cl 30 DNA-treated P⁻ JB6 Cl 30 cells yielded 40 to 50 colonies per 10⁵ cells. In contrast, transfection of Cl 30 cells with “P⁺ DNA” derived from either Cl 41 or Cl 22 yielded 200 to 500 TPA-induced colonies per 10⁵ cells, or a five- to eightfold enhancement of promotability. The enhanced promotability obtained after transfection with P⁺ DNA was stable, as judged by the retention of promotability for at least eight passages in cell lines derived from TPA-induced agar colonies. Other transfectants showed irreversible transformation by TPA, as observed in the parental P⁺ lines. When NIH 3T3 cells instead of the putative preneoplastic JB6 Cl 30 cells were used as recipients for transfection of P⁺ DNA, no evidence for acquisition of promotability was obtained. P⁻ JB6 Cl 25, like Cl 30, also permitted expression of transfected P⁺ DNA. These results suggest that sensitivity to phorbol ester promotion of transformation in JB6 cells is determined by DNA sequence(s) present in the P⁺ DNA and requires recipient cells of the appropriate phenotype for expression.     

Effects of anti-embryonal carcinoma serum on aggregation and metabolic cooperation between teratocarcinoma cells

Effects of rabbit anti-embryonal carcinoma IgG on embryonal carcinoma cells and their differentiated derivatives were studied at different levels of cell-cell interaction. Fab fragments of anti-EC IgG were found to inhibit aggregation of the majority of EC cell lines. Two, however, were insensitive. Anti-EC Fab fragments act also on the transfer of metabolites between EC cells: the rescue of HPRT^? EC cells by HPRT⁺ EC cells in selective medium is abolished. These findings are correlated with the disappearance of tight and gap junctions from the surface of EC cells (Dunia et al., 1979). The presence of the surface structure involved in the action of anti-EC Fab fragments was tested by absorption experiments followed by decompaction test on PCC4 Aza R1 cells. All EC cell lines and two embryonic cell lines—a trophectodermal and an endodermal line—were found to bear material absorbing the decompacting activity. The results are discussed in terms of state of differentiation of the cell lines and of complexity of aggregation of embyronic cells.     

A Rho GDP Dissociation Inhibitor Produced by Apoptotic T-Cells Inhibits Growth of Mycobacterium tuberculosis

In this study, we found that a subpopulation of CD4⁺CD25⁺ (85% Foxp3⁺) cells from persons with latent tuberculosis infection (LTBI) inhibits growth of M. tuberculosis (M. tb) in human monocyte-derived macrophages (MDMs). A soluble factor, Rho GDP dissociation inhibitor (D4GDI), produced by apoptotic CD4⁺CD25⁺ (85% Foxp3⁺) cells is responsible for this inhibition of M. tb growth in human macrophages and in mice. M. tb-expanded CD4⁺CD25⁺Foxp3⁺D4GDI⁺ cells do not produce IL-10, TGF-β and IFN-γ. D4GDI inhibited growth of M. tb in MDMs by enhancing production of IL-1β, TNF-α and ROS, and by increasing apoptosis of M. tb-infected MDMs. D4GDI was concentrated at the site of disease in tuberculosis patients, with higher levels detected in pleural fluid than in serum. However, in response to M. tb, PBMC from tuberculosis patients produced less D4GDI than PBMC from persons with LTBI. M. tb-expanded CD4⁺CD25⁺ (85% Foxp3⁺) cells and D4GDI induced intracellular M. tb to express the dormancy survival regulator DosR and DosR-dependent genes, suggesting that D4GDI induces a non-replicating state in the pathogen. Our study provides the first evidence that a subpopulation of CD4⁺CD25⁺ (85% Foxp3⁺) cells enhances immunity to M. tb, and that production of D4GDI by this subpopulation inhibits M. tb growth.