Entamoeba histolytica and Entamoeba dispar: prevalence infection in a rural Mexican community

The frequency of Entamoeba histolytica and Entamoeba dispar infection was analyzed in a rural community in the state of Morelos, Mexico, through PCR technique by using specie specific primer. The E. histolytica specie was detected in 33 of 290 analyzed stool samples (11.4%), E. dispar specie was observed in 21 samples (7.2%) and both species of Entamoeba were detected in seven samples (2.4%). So a higher E. histolytica than E. dispar frequency infection was detected (13.8 versus 9.6%). Even though in our design we did not considered the follow-up of included individuals, the absence of invasive amebiasis cases in the studied population during our stay in town was unexpected.     

Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

Entamoeba histolytica: alterations in EhRabB protein in a phagocytosis deficient mutant correlate with the Entamoeba dispar RabB sequence

We analyzed the expression and location of EhRabB in clone L-6, a phagocytosis-deficient mutant of Entamoeba histolytica, in comparison with the wild-type clone A. Intriguingly, trophozoites of clone L-6 express more EhRabB than those of clone A. However, the majority of EhRabB-containing vesicles remained in the cytoplasm of clone L-6 during phagocytosis. To investigate molecular alterations in EhRabB of clone L-6 we compared the EhrabB gene sequences from clones L-6 and A. We also isolated, sequenced and compared the RabB protein of Entamoeba dispar. Results showed that EhrabB gene of clone L-6 is 98.2 and 94.1% identical to rabB genes of E. dispar and clone A, respectively. The rabB genes from clone A and E. dispar have 92.2% identity. Four out of five amino acids changes in RabB proteins of clone L-6 and E. dispar are shared. These changes may alter the binding of effector proteins and the specific subcellular location of EhRabB.     

Entamoeba histolytica: fibrilar aggregates in dividing trophozoites

Entamoeba histolytica trophozoite cytokinesis is dependent upon cytoskeletal elements such as filamentous actin and myosin. Here we present confocal and transmission electron microscopy studies of this process. A sequence in the formation of the contractile ring was shown with rhodamine-phalloidine staining. Ultrastructural analysis revealed the presence of fibrilar aggregates in the cytoplasm of dividing trophozoites. Among them two filaments of different diameter were identified. These aggregates presented repeating assemblies of thin and thick filaments that in cross section revealed a muscle-like appearance. Our results suggest that these aggregates constitute the contractile ring responsible for the separation of daughter cells.     

Na+/H+ exchanger and reperfusion-induced ventricular arrhythmias in isolated perfused heart: possible role of amiloride

The roles of the Na⁺/H⁺ exchange system in the development and cessation of reperfusion induced ventricular arrhythmias were studied in the isolated perfused rat heart. The hearts were perfused in the working heart mode with modified Krebs Henseleit bicarbonate (KHB) buffer and whole heart ischemia was induced by a one-way ball valve with 330 beat/min pacing. Ischemia was continued for 15 min followed by 20 min of aerobic reperfusion (control). Amiloride (1.0mM), an inhibitor of the Na⁺/H⁺ exchange system, was added to the KHB buffer only during reperfusion (group B) or only during ischemic periods (group C). Electrocardiographic and hemodynamic parameters were monitored throughout the perfusion. Coronary effluent was collected through pulmonary artery cannulation and PO₂, PCO₂, HCO ₃ ^– and pH were measured by blood-gas analyzer.The incidence of reperfusion induced ventricular arrhythmias was 100%, 100% and 0% in control, group B and group C, respectively. The mean onset time of termination of reperfusion arrhythmias was significantly shorter in group B than in control. PCO₂ increased from 39.0±0.9 to 89.3±6.0 mmHg at the end of ischemia in control and from 40.6±0.4 to 60.5±5.8 in group C, the difference between groups was statistically significant. HCO ₃ ^– level decreased from 21.8±0.1 to 18.3±0.5 mmol/l in control, however, this decrease was significantly inhibited in group C (from 22.0±0.5 to 20.3±0.2). The increase in PCO₂ and the decrease in HCO ₃ ^– in group B were similar over time to those observed in control. The decrease in pH produced by ischemia was marked in control (from 7.35±0.01 to 6.92±0.04) and group B (from 7.34±0.01 to 6.94±0.02), whereas a decrease in pH was significantly prevented in group C (from 7.34±0.01 to 7.15±0.04). There were no significant differences in PCO₂, HCO ₃ ^– or pH among the three groups during reperfusion.These experiments provide evidence that amiloride significantly prevented the incidence of reperfusion arrhythmias when added only during ischemia and significantly terminated reperfusion arrhythmias when added only during reperfusion. Amiloride may prevent a decrease in pH, due to alterations in PCO₂ and/or HCO ₃ ^– . These changes in PCO₂ and HCO ₃ ^– might be indirectly influenced by inhibition of the Na⁺/H⁺ exchange system via Cl^–/HCO ₃ ^– exchange. The mechanism by which amiloride terminates reperfusion arrhythmias seems to involve electrophysiological effects which were not directly addressed in this experiment.     

Mitochondrial oscillation and activation of H+/cation exchange

Mitochondria incubated aerobically in the presence of tetrapropylammonium and weak acids and in the presence of trace amounts of tetraphenylboron undergo a series of damped oscillations reflecting cycles of osmotic swelling and shrinkage. The matrix volume changes are consequent to transport of tetrapropylammonium catalytically stimulated by tetraphenylboron. The amplitude and frequency of the oscillations increase with the concentration of tetrapropylammonium, as required for critical rates and extents of ion influx. Addition of bovine serum albumin abolishes both the uptake of tetrapropylammonium and the oscillations. Volume oscillations are paralleled by cyclic activation and depression of the respiratory rate. Two lines of evidence suggest that the train of damped oscillations depends on the cyclic activation of an electroneutral exchange of H⁺ with organic cations rather than on cyclic uncoupling. First, further increase of cation permeability due to a pulse of tetraphenylboron, after initiation of cation efflux, restores cation influx. Second, addition of Mg²⁺, which abolishes the oscillations, has a much more marked inhibitory effect on the process of cation efflux than on cation influx. Conversely, addition of A23187, which removes membrane-bound Mg²⁺, promotes cation efflux and thus the oscillations. It is suggested that, in the present system, stretching of the inner membrane and Mg²⁺ depletion result in activation of an electroneutral H⁺/organic cation exchange, and that cyclic activation of this reaction results in damped oscillations.     

The Na+ cycle of extreme alkalophiles: A secondary Na+/H+ antiporter and Na+/solute symporters

Extremely alkalophilic bacteria that grow optimally at pH 10.5 and above are generally aerobic bacilli that grow at mesophilic temperatures and moderate salt levels. The adaptations to alkalophily in these organisms may be distinguished from responses to combined challenges of high pH together with other stresses such as salinity or anaerobiosis. These alkalophiles all possess a simple and physiologically crucial Na⁺ cycle that accomplishes the key task of pH homeostasis. An electrogenic, secondary Na⁺/H⁺ antiporter is energized by the electrochemical proton gradient formed by the proton-pumping respiratory chain. The antiporter facilitates maintenance of a pH_in that is two or more pH units lower than pH_out at optimal pH values for growth. It also largely converts the initial electrochemical proton gradient formed by respiration into an electrochemical sodium gradient that energizes motility as well as a plethora of Na⁺/solute symporters. These symporters catalyze solute accumulation and, importantly, reentry of Na⁺. The extreme nonmarine alkalophiles exhibit no primary sodium pumping dependent upon either respiration or ATP. ATP synthesis is not part of their Na⁺ cycle. Rather, the specific details of oxidative phosphorylation in these organisms are an interesting analogue of the same process in mitochondria, and may utilize some common features to optimize energy transduction.     

The Na+/H+ antiporter of the thermohalophilic bacterium Rhodothermus marinus

In the thermohalophilic bacterium Rhodothermus marinus, the NADH:quinone oxidoreductase (complex I) is encoded by two single genes and two operons, one of which contains the genes for five complex I subunits, nqo10-nqo14, a pterin carbinolamine dehydratase, and a putative single subunit Na+/H+ antiporter. Here we report that the latter encodes indeed a functional Na+/H+ antiporter, which is able to confer resistance to Na+, but not to Li+ to an Escherichia coli strain defective in Na+/H+ antiporters. In addition, an extensive amino acid sequence comparison with several single subunit Na+/H+ antiporters from different groups, namely NhaA, NhaB, NhaC, and NhaD, suggests that this might be the first member of a new type of Na+/H+ antiporters, which we propose to call NhaE.     

K+/H+ antiport in mitochondria

Mitochondria contain a latent K⁺/H⁺ antiporter that is activated by Mg²⁺-depletion and shows optimal activity in alkaline, hypotonic suspending media. This K⁺/H⁺ antiport activity appears responsible for a respiration-dependent extrusion of endogenous K⁺, for passive swelling in K⁺ acetate and other media, for a passive exchange of matrix⁴²K⁺ against external K⁺, Na⁺, or Li⁺, and for the respiration-dependent ion extrusion and osmotic contraction of mitochondria swollen passively in K⁺ nitrate. K⁺/H⁺ antiport is inhibited by quinine and by dicyclohexylcarbodiimide when this reagent is reacted with Mg²⁺-depleted mitochondria. There is good suggestive evidence that the K⁺/H⁺ antiport may serve as the endogenous K⁺-extruding device of the mitochondrion. There is also considerable experimental support for the concept that the K⁺/H⁺ antiport is regulated to prevent futile influx-efflux cycling of K⁺. However, it is not yet clear whether such regulation depends on matrix free Mg²⁺, on membrane conformational changes, or other as yet unknown factors.     

Entamoeba histolytica: ADP-ribosylation of secreted glyceraldehyde-3-phosphate dehydrogenase

In addition to its classic glycolytic role, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated in many activities unrelated to glycolysis, such as membrane fusion, binding to host proteins and signal transduction. GAPDH can be the target of several modifications that allow incorporation to membranes and possible regulation of its activity; among these modifications is mono-ADP-ribosylation. This post-translational modification is important for the regulation of many cellular processes and is the mechanism of action of several bacterial toxins. In a previous study, we observed the extracellular ADP-ribosylation of a 37-kDa ameba protein. We report here that GAPDH and cysteine synthase A are the main ADP-ribosylated proteins in Entamoeba histolytica extracellular medium, GAPDH is secreted from ameba at 37 degrees C in a time-dependent manner, and its enzymatic activity is not inhibited by ADP-ribosylation. Extracellular GAPDH from ameba may play an important role in the survival of this human pathogen or in interaction with host molecules, as occurs in other organisms.     

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.     

Conformational changes in NhaA Na+/H+ antiporter

Abstract

Na⁺/H⁺ antiporters play a primary role in Na⁺/H⁺ homeostasis in cells and many organelles and have long been drug targets. The X-ray structure of NhaA, the main antiporter of Escherichia coli, provided structural insights into the antiport mechanism and its pH regulation and revealed a novel fold; six of the 12 TMs (Trans membrane segments) are organized in two topologically inverted repeats, each with one TM interrupted by an extended chain creating a unique electrostatic environment in the middle of the membrane at the cation binding site. Remarkably, inverted repeats containing interrupted helices with similar functional implications have since been observed in structures of other bacterial secondary transporters with almost no sequence homology. Finally, the structure reveals that NhaA is organized into two functional regions: a ‘pH sensor' – a cluster of amino acyl side chains that are involved in pH regulation; and a catalytic region that is 9 Å removed from the pH sensor. Alternative accessibility of the binding site to either side of the membrane, i.e., functional-dynamics, is the essence of secondary transport mechanism. Because NhaA is tightly pH regulated, structures of the pH-activated and ligand-activated NhaA conformations are needed to identify its functional-dynamics. However, as these are static snapshots of a dynamic protein, the dynamics of the protein both in vitro and in situ in the membrane are also required as reviewed here in detail. The results reveal two different conformational changes characterizing NhaA: One is pH-induced for NhaA activation; the other is ligand-induced for antiport activity.     

H+/Ca2+ exchange in rabbit renal cortical endosomes

Summary We have examined the effect of second messengers on ATP-driven H⁺ transport in an H⁺ ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1mm) had no effect on H⁺ transport. Acridine orange fluorescence in the presence of 0.5mm Ca²⁺ (+1mm EGTA) was 19±6% of control. Inhibition of ATP-driven H⁺ transport by Ca²⁺ was concentration dependent; 0.25 and 0.5mm Ca²⁺ (+1mm EGTA) inhibited acridine orange fluorescence by 50 and 80%, respectively. Ca²⁺ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca²⁺ did not affect ATP hydrolysis. ATP-dependent Br^– uptake was virtually unchanged in the presence of 0.5mm Ca²⁺ (+1mm EGTA). These vesicles were also shown to transport Ca²⁺ in an ATP-dependent mode. Inositol 1, 4, 5-trisphosphate had no effect on ATP-dependent Ca²⁺ uptake. These results are consistent with the co-existence of an H⁺ ATPase and an H⁺/Ca²⁺ exchanger on these endosomes, the latter transport system using the H⁺ gradient to energize Ca²⁺ uptake. Attempts to demonstrate an H⁺/Ca²⁺ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca²⁺ uptake could be demonstrated. A Ca²⁺-dependent increase in H⁺ permeability and an ATP-dependent Ca²⁺ uptake might have important implications for the regulation of vacuolar H⁺ ATPase activity as well as the homeostasis of cytosolic Ca²⁺ concentration.     

Species- and strain-specific probes derived from repetitive DNA for distinguishing Entamoeba histolytica and Entamoeba dispar

Entamoeba histolytica and Entamoeba dispar are two morphologically indistinguishable species that are found in the human gut. Of the two, E. histolytica is considered to be pathogenic while E. dispar is nonpathogenic. To generate molecular probes to detect and distinguish between the two species, we utilized repeat sequences present in Entamoeba genome. We have developed probes and primers from rDNA episomes, and unidentified Entamoeba EST1 repeat for this purpose, and used them for dot blot hybridization and PCR amplification. To investigate the possible existence of invasive and noninvasive strains of E. histolytica, the ability to differentiate individual isolates is necessary. For this purpose, we have utilized a modification of the AFLP procedure called 'Transposon display,' which generates and displays large number of genomic bands associated with a transposon. We have used the abundant retrotransposon, EhSINE1, for this purpose,and demonstrated its potential as a marker to study strain variation in E. histolytica. This technique could suitably be employed in carrying out significant molecular epidemiological studies and large-scale typing of this parasite.     

Trichinella spiralis: infection reduces airway allergic inflammation in mice

In an effort to define the mechanism underlying the host immune downregulation inherent to Trichinella spiralis infection, we compared the levels of Th1, Th2, and regulatory cytokines and CD4⁺CD25⁺ forkhead box P3 (FoxP3)⁺ T (T_reg) cell recruitment, as well as cellular pathology in the airway between T. spiralis infected and uninfected asthma-induced mice. After the induction of allergic airway inflammation, we noted influxes of inflammatory cells into the peribronchial tree. However, in the T. spiralis infection groups, cellular infiltration was minimal around the bronchial tree, with only a smattering of inflammatory cells. In the OVA-challenged group after T. spiralis infection, the numbers of macrophages and eosinophils in the bronchial alveolar lavage fluid were reduced by 23% and 52%, respectively, as compared to those of the OVA-challenged group. Airway hyperresponsiveness of OVA-challenged mice after T. spiralis infection was significantly suppressed as compared to the OVA-only challenged mice. The T. spiralis-infected mice exhibited a significant reduction in IL-5 concentrations relative to that noted in the OVA-challenged group (p < 0.01). Nevertheless, the regulatory cytokines IL-10 and TGF-β levels were increased significantly as the result of T. spiralis infection, and we verified the recruitment of T_reg cells in lung draining lymph nodes via T. spiralis infection. Therefore, T_reg cells, which were recruited by T. spiralis infection, might ameliorate lung function and reduce allergic airway inflammation.     

Codon Usage in Pathogenic Entamoeba histolytica

Summary An analysis of 4680 codons expressed by pathogenic Entamoeba histolytica showed the A+U content of coding sequences to be 67%. The preference for A+U resulted in an unusual codon usage with an A+U content of 84% in the third codon position. The data show a remarkable similarity to those obtained for Plasmodium falciparum.     

Respiration-dependent contraction of swollen heart mitochondria: Participation of the K+/H+ antiporter

Respiration-dependent contraction of heart mitochondria swollen passively in K⁺ nitrate is activated by the ionophore A23187 and inhibited by Mg²⁺. Ion extrusion and osmotic contraction under these conditions are strongly inhibited by quinine, a known inhibitor of the mitochondrial K⁺/H⁺ antiporter, as measured in other systems. The inhibition by quinine is relieved by the exogenous antiporter nigericin. Respiration-dependent contraction is also inhibited by dicyclohexylcarbodiimide (DCCD) when reacted under conditions known to inhibit K⁺/H⁺ antiport (Martinet al., J. Biol. Chem. 259, 2062–2065, 1984). These studies strongly support the concept that K⁺ is extruded from the matrix by the endogenous K⁺/H⁺ antiporter and that inhibition of this component by quinine or DCCD inhibits respiration-dependent contraction. The extrusion of K⁺ nitrate is accompanied by a respiration-dependent efflux of a considerable portion of the endogenous Mg²⁺. This Mg²⁺ efflux does not occur in the presence of nigericin or when the mitochondrial Na⁺/H⁺ antiporter is active. Mg²⁺ efflux may take place on the K⁺/H⁺ antiporter. DCCD, reacted under conditions that do not result in inhibition of the K⁺/H⁺ antiporter, blocks a monovalent cation uniport pathway. This uniport contributes to futile cation cycling at elevated pH, and its inhibition by DCCD stimulates respiration-dependent contraction.     

ATP Dependence of Na+/H+ Exchange : Nucleotide Specificity and Assessment of the Role of Phospholipids

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na⁺/H⁺ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pH_i) by microfluorimetry. Na⁺/H⁺ exchange activity was measured as the Na⁺-driven pH_i recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na⁺/H⁺ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-d-glucose and oligomycin. In cells dialyzed in the presence of ATP, no “run-down” was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at ∼5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na⁺/H⁺ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca²⁺ exchanger, this requirement has been attributed to an aminophospholipid translocase, or “flippase.” The involvement of this enzyme in Na⁺/H⁺ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na⁺/H⁺ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATPγS were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTPγS was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg²⁺ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the γ-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.     

T cell precursor frequency differentially affects CTL responses under different immune conditions

Generation of effective CTL responses is the goal of many vaccination protocols. However, to what extant T cell precursor frequencies will generate a CD8⁺ CTL response has not been elucidated properly. In this study, we employed a model system, in which naive CD4⁺ and CD8⁺ T cells derived from ovalbumin (OVA)-specific TCR transgenic OT II and OT I mice were used for adoptive transfer into wild-type, Ia^b−/− gene knockout and transgenic RIP-mOVA mice, and assessed OVA-pulsed DC (DC_OVA)-stimulated CD8⁺ CTL responses in these mice. We demonstrated that (i) a critical threshold exists above which T cells precursor frequency cannot enhance the CTL responses in wild-type C57BL/6 mice, (ii) increasing CD8⁺ T cell precursors is required to generate CTL responses but with functional memory defect in absence of CD4⁺ T cell help, and (iii) increasing CD4⁺ and CD8⁺ T cell precursors overcomes immune suppression to DC_OVA-stimulated CD8⁺ CTL responses in transgenic RIP-mOVA mice with OVA-specific self immune tolerance. Taken together, these findings may have important implications for optimizing immunotherapy against cancer.