Interpretation of 51Cr-release data: a kinetic analysis.

An analysis of 51Cr-release data from cell-mediated cytotoxicity assays, plotted in the form of effector cell dilution curves, is analyzed in detail in terms of a saturation kinetics model for effector-target interaction. The effect of nonimmune (bystander) cells in the effector population is particularly examined. The theoretical development of the saturation model predicts that, at low concentrations of target cells, increasing proportions of bystander cells should broaden the range of effector cell concentration required to achieve a given increment of target cell lysis, but that at high target cell concentration this effect should disappear. Experimental validation of this prediction is presented.     

Killing of targets by CD8 T cells in the mouse spleen follows the law of mass action

It has been difficult to correlate the quality of CD8 T cell responses with protection against viral infections. To investigate the relationship between efficacy and magnitude of T cell responses, we quantify the rate at which individual CD8 effector and memory T cells kill target cells in the mouse spleen. Using mathematical modeling, we analyze recent data on the loss of target cells pulsed with three different peptides from the mouse lymphocytic choriomeningitis virus (LCMV) in mouse spleens with varying numbers of epitope-specific CD8 T cells. We find that the killing of targets follows the law of mass-action, i.e., the death rate of individual target cells remains proportional to the frequency (or the total number) of specific CD8 T cells in the spleen despite the fact that effector cell densities and effector to target ratios vary about a 1000-fold. The killing rate of LCMV-specific CD8 T cells is largely independent of T cell specificity and differentiation stage. Our results thus allow one to calculate the critical T cell concentration at which growth of a virus with a given replication rate can be prevented from the start of infection by memory CD8 T cell response.     

THE KINETICS OF 51,Cr RELEASE FROM TARGET CELLS IN CELL MEDIATED CYTOTOXICITY AND THE RELATIONSHIP TO THE KINETICS OF KILLING

The kinetics of T-cell mediated cytotoxicity (CMC) have been re-examined. It is clear that per cent ⁵¹Cr release is linearly related to effector cell number and not to its log as often suggested. Data are presented showing that killing can occur within 1 min of lymphocyte-target cell contact and that ⁵¹Cr is released rapidly from killed cells. Inactivation experiments aimed to stop ongoing cytolysis indicate that all target cells are brought into contact with effector lymphocytes within 45 min. At a ratio of 50:1 the kinetics of contact resemble saturation kinetics, indicating a number of effector cells similar to or in excess of the number of target cells (that is, at least 2 % of total lymphocytes). Although CMC is causally related to cell contact, it is chronometrically unrelated and occurs as a random reaction (that is it can occur immediately or up to hours later than contact). The mean time depends on the number of effector cells. The killing reaction is temperature dependent and proportional to the number of effector cells but does not depend on the intact effector cell. An intact effector cell is only required to bring about contact. It is suggested that this final cytolytic event is brought about by the target cell itself, perhaps as a result of attempts to free itself from the attached lymphocyte.     

Transport-limited growth in the chemostat and its competitive inhibition; A theoretical treatment

Summary An equation expressing the specific growth rate of heterotrophic cell populations in terms of yield factor and transport rate is proposed. From this equation expressions are derived for the specific growth rate when the transport of the energy source is growth0limiting. These expressions are applied to cell population growth in the chemostat limited by the transport of the energy source or of other substrates and simple mathematical tools are provided for obtaining estimates of the transport parameters. An equation is derived which predicts that at constant dilution rate in the chemostat the concentration of any substrate (whether or not the source of energy) the transport of which is growth limiting, is a linear function of the concentration of a competitive inhibitor of its transport. With this equation estimates of the Michaelis constants of competitive transport inhibitors can be obtained. The growth rate equation of Monod (1942) is discussed.     

The functional profile of primary human antiviral CD8+ T cell effector activity is dictated by cognate peptide concentration

Antiviral CD8(+) T cells can elaborate at least two effector functions, cytokine production and cytotoxicity. Which effector function is elaborated can determine whether the CD8(+) T cell response is primarily inflammatory (cytokine producing) or antiviral (cytotoxic). In this study we demonstrate that cytotoxicity can be triggered at peptide concentrations 10- to 100-fold less than those required for cytokine production in primary HIV- and CMV-specific human CD8(+) T cells. Cytolytic granule exocytosis occurs at peptide concentrations insufficient to cause substantial TCR down-regulation, providing a mechanism by which a CD8(+) T cell could engage and lyse multiple target cells. TCR sequence analysis of virus-specific cells shows that individual T cell clones can degranulate or degranulate and produce cytokine depending on the Ag concentration, indicating that response heterogeneity exists within individual CD8(+) T cell clonotypes. Thus, antiviral CD8(+) T cell effector function is determined primarily by Ag concentration and is not an inherent characteristic of a virus-specific CD8(+) T cell clonotype or the virus to which the response is generated. The inherent ability of viruses to induce high or low Ag states may be the primary determinant of the cytokine vs cytolytic nature of the virus-specific CD8(+) T cell response.     

Structure of a peptidoglycan amidase effector targeted to Gram-negative bacteria by the type VI secretion system

The target range of a bacterial secretion system can be defined by effector substrate specificity or by the efficacy of effector delivery. Here, we report the crystal structure of Tse1, a type VI secretion (T6S) bacteriolytic amidase effector from Pseudomonas aeruginosa. Consistent with its role as a toxin, Tse1 has a more accessible active site than related housekeeping enzymes. The activity of Tse1 against isolated peptidoglycan shows its capacity to act broadly against Gram-negative bacteria and even certain Gram-positive species. Studies with intact cells indicate that Gram-positive bacteria can remain vulnerable to Tse1 despite cell wall modifications. However, interbacterial competition studies demonstrate that Tse1-dependent lysis is restricted to Gram-negative targets. We propose that the previously observed specificity for T6S against Gram-negative bacteria is a consequence of high local effector concentration achieved by T6S-dependent targeting to its site of action rather than inherent effector substrate specificity.     

Regulation of human natural killing by levamisole

Summary The effect of levamisole on human natural killing (NK) has been studied. In short-term chromium release assays, levamisole at a concentration of 10^–3 M was inhibitory to NK when present in the assays. Pretreatment of NK effector cells and K562 target cells with levamisole separately indicated that the effect was on effector cell activity and was not due to any change in target cell susceptibility. Inactivation of the effector cells required greater than 4 h pretreatment with levamisole if NK activity was subsequently tested in the absence of the drug. Pretreatment with levamisole for up to 19 h had no effect on the lymphocyte proliferative response to phytohemagglutinin (PHA). NK activity of drug-inactivated effector cells recovered after further incubation in levamisole-free medium. Levamisole at 10^–4 M or less had no effect on NK either by pretreatment or by its presence in the NK assays.     

Antibody-dependent cell-mediated cytotoxicity in humans. II. Energy requirement.

Antibody-dependent cytotoxicity mediated by human peripheral lymphocytes is an active energy-requiring phenomenon. In this study the relative importance of glycolysis and respiration was analyzed, by measuring the effect of various metabolic inhibitors (sodium azide, antimycin A, 2-deoxy-D-glucose, iodoacetate), alone or in combination, on effector cell efficiency and intracellular ATP level. The inhibition of both aerobic and anaerobic energy production in the effector cells completely abolished cytotoxicity. An inhibition of 50% was observed when the intracellular ATP concentration was decreased to levels corresponding to 30 to 50% of those in the untreated controls.     

The atypical velocity response by pyruvate carboxylase to increasing concentrations of acetyl-coenzyme A

An investigation was made of the interaction of pyruvate carboxylase with its allosteric effector, acetyl-CoA, and the velocity profile of the deacylation of acetyl-CoA as a function of acetyl-CoA concentration indicated that this ligand does not bind to this enzyme in a positive homotropic co-operative manner. An examination was therefore made of the factors that contribute to the sigmoidicity of the rate curves obtained for pyruvate carboxylation with various concentrations of acetyl-CoA. Hill coefficients for acetyl-CoA obtained with both sheep and chicken liver pyruvate carboxylases were found to be dependent on the fixed pyruvate concentration used in the assay solution. Thus, by varying the acetyl-CoA concentration, the degree of saturation of the enzyme by pyruvate was also changed. A further consequence of non-saturating concentrations of pyruvate was that the non-productive hydrolysis of the enzyme- carboxybiotin complex increased, resulting in an under-estimate of the reaction velocity measured by oxaloacetate formation. Another factor contributing to the sigmoidicity is that, at non-saturating concentrations of acetyl-CoA, the enzyme undergoes inactivation upon dilution to low protein concentrations, again resulting in an under-estimate of the reaction velocity. Under conditions where none of the above factors was operating and the only effect of varying acetyl-CoA concentrations was to alter the proportion of the enzyme catalysing the carboxylation reaction at acetyl-CoA-dependent and -independent rates, the sigmoidicity of the acetyl-CoA velocity profile was completely eliminated.     

Inhibition of natural killer cell activity of human lymphocytes by eicosapentaenoic acid

The effect of eicosapentaenoic acid (EPA) on natural killer (NK) cell activity of human lymphocytes was examined. The addition of an emulsion of trieicosapentaenoyl-glycerol (EPA-TG) emulsified with purified phosphatidylcholine from krill to a cytotoxicity assay system resulted in a marked depression of NK activity. The inhibition was proportional to the concentration of EPA-TG emulsion, and was observed as early as the first one hour of incubation at various effector to target cell ratios. Pretreatment of effector cells with EPA-TG emulsion resulted in significant suppression of their NK activity. Inhibition of cytotoxicity was not due to direct toxicity to effector cells or decreased target cell binding. These results indicate that EPA is a potent inhibitor of NK activity in vitro.     

A reaction cell for simultaneous measurements of fluorescence and absorption in a hemoglobin solution at various oxygen pressures

An apparatus was constructed to carry out measurements of fluorescence, optical absorption and oxygen partial pressure in a hemoglobin or other solution simultaneously, and its performance was examined. This apparatus has a rhombiform optical cell in place of the usual square optical cell used in commercially available spectrofluorometers. Fluorescence emitted at the region near the cell surface in the solution could be detected satisfactorily and easily even if the solution had strong light absorption bands at both the excitation and the emission wavelengths in the presence of high concentrations of a chromophore. This apparatus was particularly effective for studies on the interactions of a fluorescent allosteric effector with hemoglobin at various degrees of deoxygenation. Consequently, it was proved experimentally that the fluorescence of β-naphthyl triphosphate bound to hemoglobin is completely quenched. Moreover, simultaneous and continuous measurements of the oxygen-binding equilibrium of hemoglobin and the allosteric effector-binding to hemoglobin as a function of oxygen partial pressure could be satisfactorily carried out, and it is confirmed that β-naphthyl triphosphate binds not only to deoxyhemoglobin but also to fully oxygenated hemoglobin and lowers strongly the oxygen affinity of hemoglobin as an allosteric effector.     

Role of the renin-angiotensin system in tubuloglomerular feedback

The link between the renal tubule and glomerular vasculature comprised of the juxtaglomerular apparatus appears to serve two functions: the regulation of filtration rate and of renin secretion. Elevation of macula densa NaCl concentration stimulates a vasoconstrictor response, which results in a fall in filtration rate, a response that has been termed tubuloglomerular feedback (TGF). Simultaneously, renin secretion is suppressed. The two responses appear to be initiated by a furosemide-sensitive transport step probably located in the macula densa. Both show a pattern of anion specificity identical to Na/K/Cl cotransport mechanisms. An increase in intracellular calcium in the effector cells, the vascular smooth muscle, and the renin-containing granular cells is a likely effector mechanism for both reactions. Angiotensin probably does not mediate the vasoconstrictive feedback response, because changes in local (intracellular) angiotensin concentration would have to be opposite from systemic changes. However, acute changes in angiotensin levels appear to be an important modulator of the magnitude of the TGF response.     

A colorimetric assay for studying effector secretion through the bacterial type III secretion system

We have devised a colorimetric method that monitors secretion of effector proteins into host cytoplasm through the bacterial type III secretion machinery. Here we used constructs of effectors fused with Bordetella adenylate cyclase as a reporter, but evaluated the effector translocation by quantifying cell viability, rather than by measuring the intracellular cAMP concentration. This is based on our findings that cells infected by a secretion-competent bacterium expressing the fusion protein lost their viability under our experimental conditions. Cell death was quantified using commercially available reagents and basic research equipment. An observation that cell death was potentiated when the infected cells were treated with 2-deoxyglucose and sodium azide suggests that the depletion of intracellular ATP is partly involved in the process. Using enteropathogenic Escherichia coli, we demonstrated that the method was applicable to at least three effectors of bacteria, Tir, EspF, and Map, and was useful for studying a secretion signal sequence for Tir. This technically simple and inexpensive method is a good alternative to the existing procedure for studying the mechanism by which effectors are secreted through the type III secretion system in a high-throughput format.     

Two distinct stages in the transition from naive CD4 T cells to effectors, early antigen-dependent and late cytokine-driven expansion and differentiation

Efficient peptide presentation by professional APC to naive and effector CD4 T cells in vitro is limited to the first 1-2 days of culture, but is nonetheless optimum for effector expansion and cytokine production. In fact, prolonging Ag presentation leads to high levels of T cell death, decreased effector expansion, and decreased cytokine production by recovered effectors. Despite the absence of Ag presentation beyond day 2, T cell division continues at a constant rate throughout the 4-day culture. The Ag-independent later stage depends on the presence of IL-2, and we conclude optimum effector generation depends on an initial 2 days of TCR stimulation followed by an additional 2 days of Ag-independent, cytokine driven T cell expansion and differentiation.     

Antibody-dependent cytolytically active human leukocytes: an analysis of inactivation following in vitro interaction with antibody-coated target cells.

A leukocyte population consisting of approximately 85% lymphocytes, prepared from human peripheral blood by centrifugation through a Ficoll-Hypaque gradient, was studied for its capacity to destroy antibody-coated human liver (Chang) cells in vitro. Cytolysis was a rapid event: increased ionic flux (86Rb) from the target cell occurred within 10 min of the addition of effector cells. Kinetic analysis of target cell destruction (51 Cr release) was compatible with a "one hit" hypothesis, thereby indicating that cytolysis resulted from a single collision was an effector cell. The initial rate of cytolysis was linear and related to the number of leukocytes added, but lysis at all of the leukocyte to target cell ratios tested ceased after 5 hr. The number of target cells killed at that time was directly proportional to the number of leukocytes added. While the lytic capacity of the effector population was totally depleted after incubation with antibody-coated target cells, cytotoxicity was not affected by co-culturing leukocytes with Chang cells treated with pre-immune serum. The cytotoxic effector cells functioning in this antibody-dependent lytic system are thus to be contrasted with killer T cells, whose lytic activity is not compromised by interaction with homologous target cells. It was estimated that approximately 4% of the leukocyte population employed could kill antibody-coated Chang cells, a figure consistent with the estimated frequency of "null" cells within human peripheral lymphocytes.     

Effect of histamine and histamine antagonists on natural and antibody-dependent cellular cytotoxicity of human lymphocytes in vitro

The in vitro effect of histamine and its antagonists, cimetidine and clemastine fumarate, on natural killer (NK) and antibody-dependent cellular Cytotoxicity (ADCC) activities of human lymphocytes was investigated. The histamine 1 (H1) antagonist, clemastine fumarate, and the histamine 2 (H2) antagonist, cimetidine, but not histamine alone, inhibited the NK and ADCC activities of lymphocytes when added directly to the mixture of effector and target cells in a ⁵¹Cr-release assay. This inhibition was proportional to the concentration of drugs added and was observed at various effector to target ratios against several targets. H1 and H2 antagonists also inhibited NK activities of T cells as well as Percoll-separated, NK-enriched effector cells. The inhibition was significantly reversed by histamine. In target binding assays, clemastine fumarate and cimetidine also decreased the target binding capacity of effector lymphocytes. Further, PBL precultured with histamine (10^?3–10^?4M) for 24 hr showed a significant decrease in their NK and ADCC activities. In coculture experiments, PBL precultured with histamine suppressed the NK activity of normal autologous effector lymphocytes. PBL precultured with histamine showed an increased number of OKT8⁺ cells, as estimated using monoclonal antibodies. The suppression of Cytotoxicity was not due to either direct toxicity, steric hindrance, crowding, or cell death, but by functionally viable suppressor cells. An immunoregulatory role for histamine in NK and ADCC reactions is proposed.     

Population dynamics of mitochondria. I. A model for the role to ACTH in the degradation of adrenocortical mitochondria

An allosteric substance has been supposed to be present in the adrenocortical cell and to be involved in the degradation of the adrenocortical mitochondria only when it is present in the cytoplasm as a free form. An allosteric effector has also been assumed to be synthesized in the adrenal cortex strongly depending on the ACTH supply. The allosteric effector combines hypothetically with the allosteric substance to form an association product. In its bound form, the allosteric substance is assumed to be inactive in the degradation reaction of mitochondria. With these assumptions a differential equation has been obtained to describe the decay process of those mitochondria. An algorithm has been developed to compute the dynamical fate of the mitochondria in a simple, iterative way. Experimental results on the mitochondrial decay in the rat adrenal cortex after hypophysectomy have been fitted to the differential equation in a satisfactory manner. It has been stressed that the present hypothesis constitutes in its essence a new working hypothesis on the maintenance of adrenocortical mitochondria under normal physiological conditions.     

Preparation and characterization of a DOTA-lysine-biotin conjugate as an effector molecule for pretargeted radionuclide therapy

Pretargeted radionuclide therapy depends on the establishment of a high concentration of secondary binding sites at a tumor to which low-molecular weight radiolabeled effector molecules can be directed. This study describes the simple synthesis of an effector molecule and its subsequent characterization to determine the extent to which it complied with the ideal requirements of such a compound. (Epsilon)-DOTA-(alpha)-biotinamidolysine (DLB) was synthesized in high yield and purity using conventional SPPS methodology. High radiochemical purities were obtained when labeled with several potentially useful radionuclides. The radiolabeled analogue bound to streptavidin efficiently with a stoichiometry similar to that of native biotin and showed high stability in serum and upon challenge with acid conditions. Biodistribution studies in normal animals showed a rapid rate of clearance from the blood and low retention of radioactivity by normal tissues. This design of effector molecule therefore shows promise for further pretargeted radionuclide therapy studies.     

Cell growth optimization in microcarrier culture

Three monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was DEAE-Sephandex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute of Technology. The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue culture medium and a cell inoculum of 20,000 cells/cm2 of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0 mg/ml, respectively. In terms of cell yield per milliliter of tissue culture medium, the microcarrier culture was superior to roller bottle and stationary cultures. An advantage of the microcarrier culture system is its suitability for scale up into large volume production units.     

Adoptive transfer of experimental allergic encephalomyelitis: conditions influencing memory and effector cell development.

The cellular transfer of clinical experimental allergic encephalomyelitis (EAE) with immune spleen cells is only accomplished following lymphoid cell stimulation during an intervening in vitro culture activation period. Recipients of these cells recover from the ensuing adoptively transferred paralytic episode and subsequently respond to active challenge with myelin basic protein (BP)-CFA in an accelerated time frame consistent with the presence of memory cells in the initial cell transfer inoculum. We have found that the addition of anti-CD4 antibody or dexamethasone during the activation period inhibits the development of the transfer active EAE effector cell subpopulation, but does not alter the in vitro development and subsequent expression of the BP-specific memory cell subpopulation. Additional experiments also suggest the development of memory cells in the absence of effector cell activity. PMA + ionomycin when used as a stimulus during the culture activation period leads to effector and memory cell development. The stimulation response is dose dependent, in that a reduced concentration of PMA + ionomycin does not lead to EAE effector cell development; however, at these reduced levels of PMA + ionomycin, memory cell development still occurred. Additional evidence which supports the concept of independent development of memory cells and effector cells was obtained with a BP-specific cell line. Following recovery from cell line-mediated clinical EAE, as well as following adoptive transfer of the cell lines in the precursor stage, cell recipients did not develop an early onset of active EAE when subsequently immunized with BP-CFA. Thus the BP-specific T-cell line appears to contain the precursors of the effector cell subpopulation but does not appear to contain the BP memory cell subpopulation. Collectively these observations suggest the existence of distinct T-cell subsets or pathways of development that are followed during the response to BP as measured by the development of clinical EAE.