Biomedical subjects

S S Patel

Publications and source records attributed to S S Patel.

At least 73 records · Page 4Linked to original sources

PubMed1994

The K318A mutant of bacteriophage T7 DNA primase-helicase protein is deficient in helicase but not primase activity and inhibits primase-helicase protein wild-type activities by heterooligomer formation.

Lysine 318 in the conserved sequence SXXXGXGKS of bacteriophage T7 gene 4A' protein was mutated to an alanine to understand the effect of this substitution on the helicase and primase activities. The dTTPase activity of 4A'/K318A mutant protein was much lower than that of 4A', and both Km and kcat values were affected. The Km of the mutant protein was 3-5-fold higher, and the kcat was about 100-fold lower, than that of 4A'. The mutation did not affect the ability of 4A'/K318A to assemble into hexamers or bind DNA in the presence of MgdTTP. Interestingly, the mutant protein does not bind DNA in the presence of MgdTMP-PCP. The reduced dTTPase activity, however, decreased the helicase activity of the mutant protein to an undetectable level, whereas its primase activity was only 1.5-2.5-fold lower. When 4A'/K318A mutant protein was mixed with 4A', heterooligomers were formed and the helicase and the DNA-dependent dTTPase activities of 4A' were inhibited, but the DNA-independent activity actually increased. The extent of decrease in activities upon heterooligomer formation depended both on the length of time 4A' and 4A'/K318A proteins were incubated and on the concentration of the mutant protein. In addition, the decrease in the dTTPase activity was observed only when the two proteins were incubated in the absence of MgdTTP and DNA, conditions under which both proteins form unstable hexamers. Even though 4A'/K318A does not bind a 30-mer DNA in the presence of MgdTMP-PCP, heterooligomers were capable of binding DNA with the same stoichiometry as 4A'. Protein-DNA cross-linking experiments with (dT)30 and poly(5-BrdU) showed that DNA interacts with five and perhaps all six subunits of 4A'. Therefore, unless heterooligomer restores the ability of the mutant protein to bind DNA in the presence of MgdTMP-PCP, these results suggest that the DNA can bind 4A' by interacting with a few subunits. However, a fully active hexamer is required for both the helicase and the single-stranded M13 DNA-dependent dTTPase activities.

Amino Acid Sequence↗

PubMed1994

Thermal inactivation of gamma-glutamyltranspeptidase and Enterococcus faecium in milk-based systems.

Untreated whole milk, skim milk, sweetened milks, sweetened and unsweetened creams and ice-cream mixes were preincubated with a culture of Enterococcus faecium, then subjected to heat treatment in a pilot-scale plate heat exchanger using a hold of 15 s at 76 degrees C. The liquids were examined for gamma-glutamyltranspeptidase (GGTP) activity and total streptococcal count before and after heat treatment and the results plotted against water activity (aW). There was a good correlation between reduction in GGTP activity, destruction of streptococci and aW, demonstrating the potential of the enzymic assay for assessing the severity of HTST heat treatments above the minimum for pasteurization.

Animals↗

PubMed1993

Interactions of bacteriophage T7 DNA primase/helicase protein with single-stranded and double-stranded DNAs.

Protein-DNA interactions of bacteriophage T7 DNA primase/helicase protein 4A' with small synthetic oligodeoxynucleotides were investigated using a 20-base-paired hairpin duplex, and 10-, 30-, and 60-base-long single-stranded DNA. The effect of nucleotide cofactors on DNA binding was examined using membrane binding assays which showed that 4A' binds DNA optimally only in the presence of MgdTMP-PCP, the nonhydrolyzable analog of dTTP. About 20% of single-stranded DNA binding was observed in the presence of MgdTDP, but none was detectable in the absence of nucleotides. Native polyacrylamide gel electrophoresis showed that the DNAs bind predominantly to the hexameric form of 4A'. Larger oligomers of 4A' can bind DNA, but no DNA binding was observed to species smaller than the hexamer. Quantitative equilibrium binding studies at increasing 4A' concentrations and at increasing DNA concentrations showed tight binding of one 10-mer or 30-mer per hexamer. The 4A' hexamer can bind a second strand of DNA, but with a 50-fold weaker affinity than the first strand. The 60-mer showed tight binding to two 4A' hexamers, suggesting that a hexamer may interact with only 30-40 bases of single-stranded DNA. This was corroborated by nuclease protection experiments where the smallest length of DNA protected by 4A' or 4B protein was found to be about 30 bases. Equilibrium binding studies and competitive DNA binding data are consistent with a weaker affinity of 4A' for the duplex DNA. Only 20-25% of duplex DNA binding was observed at increasing 4A' protein in the presence of MgdTMP-PCP. About four duplex DNAs can bind each 4A' hexamer at increasing DNA concentrations, but their weaker binding was evident from their facile dissociation from 4A' in the presence of competing single-stranded DNA.

Bacteriophage T7↗

PubMed1993

Oligomeric structure of bacteriophage T7 DNA primase/helicase proteins.

The oligomeric structure of bacteriophage T7 gene 4 helicase/primase proteins was investigated using protein cross-linking and high pressure gel-filtration chromatography. Studies were carried out with both 4A' and 4B proteins. 4A' is a M64L mutant of 4A which has similar helicase and primase activities as the wild-type mixture of 4A and 4B proteins (Patel, S. S., Rosenberg, A. H., Studier, F. W., and Johnson, K. A. (1992) J. Biol. Chem. 267, 15013-15021), and 4B is the smaller protein which has only helicase activity. Chemical cross-linking of 4A' and 4B proteins with dimethyl suberimidate resulted in cross-linked species ranging from dimers to hexamers and beyond. The cross-linking time course, however, indicated that hexamers were the predominant species to accumulate in both 4A' and 4B proteins. The effect of MgNTP and DNA binding on oligomerization of the gene 4 proteins was investigated using high pressure gel-filtration chromatography at increasing protein concentrations. In the absence of added ligands, close to 100 microM protein concentrations were required to form stable oligomers beyond dimers. However, in the presence of Mg-beta, gamma-methylene deoxythymidine triphosphate (nonhydrolyzable analog of dTTP), 4A' and 4B protein assembled into stable hexamers at protein concentrations less than 8 microM. Addition of single-stranded DNA further stabilized the hexamer structure. Therefore, in the presence of a 60-nucleotide-long single-stranded DNA, hexamers were observed at protein concentrations as low as 0.2 microM. Nuclease protection experiments indicated that the 4A' and 4B hexamers protect about 60-65 bases of single-stranded DNA.

Bacteriophage T7↗

PubMed1993

Stimulus-dependent modulation of human B cell function by T cell clones.

T cells exert both positive and negative regulatory effects on B cell function. To determine whether the nature of the stimulus could alter the immunoregulatory effects of T cells, the capacity of a battery of human T cell clones to modulate B cell function after stimulation with either pokeweed mitogen or a mAb to the CD3 molecular complex was examined. It was observed that most clones induced B cell differentiation when stimulated with immobilized mAb to CD3 (64.1) regardless of their phenotype. Moreover, the majority of clones (42 of 51) augmented the generation of immunoglobulin-secreting cells (ISC) supported by anti-CD3-stimulated blood CD4+ T cells. By contrast, none of the clones induced B cell differentiation when stimulated with PWM and 48 of 51 clones suppressed the generation of ISC induced by blood CD4+ T cells. This suppression could not be accounted for by the depletion of essential molecules or factors or by secretion of suppressive factors. Suppressive activity of clones did not correlate with the CD4 or CD8 phenotype and was not overcome by the addition of supernatants generated from mitogen-stimulated T cells or recombinant IL-2. Suppression by most clones, however, was abrogated when the clones were treated with mitomycin C or irradiated. A number of suppressive mechanisms by individual PWM-stimulated clones was identified, including direct inhibition of B cell function by cytotoxic and non-lytic means and suppression of helper T cell function. The failure of anti-CD3-stimulated clones to suppress the differentiation of B cells appeared to reflect the capacity of this stimulus to induce apoptosis by the clones after initial activation. These results indicate that T cell clones can provide help for B cell differentiation or can suppress B cell function by a variety of mechanisms depending upon the mode of stimulation.

Animals↗

PubMed1992

Cloning and expression of gene 4 of bacteriophage T7 and creation and analysis of T7 mutants lacking the 4A primase/helicase or the 4B helicase.

T7 gene 4, which is required for DNA replication, specifies two proteins whose coding sequences overlap in the same reading frame: the 4A protein, a 566-amino acid primase/helicase, and the 4B protein, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein. To study better the individual functions of these two overlapping proteins, we made clones that express both 4A and 4B proteins, only 4B protein, or only what we refer to as the 4A' protein, in which methionine 64 is replaced by leucine, thereby eliminating the 4B initiation codon. These clones provide considerably more gene 4 protein for biochemical analysis than do infected cells. They can also be used to isolate and propagate T7 gene 4 deletion mutants, and we have made T7 mutants which lack all gene 4 coding sequences, or which express 4A' protein but no 4B protein, or 4B protein but no 4A protein. Analysis of these phage mutants shows that 4A' protein without any 4B protein can support essentially normal replication and growth, whereas 4B protein without any 4A protein supports little replication or growth. Apparently, the primase activity of the 4A protein is essential for replication, but the 4B protein is dispensable, presumably because the 4A protein also supplies helicase activity. The mutation at amino acid 64 of 4A' appears to have little effect on 4A function. The rate of replication during normal T7 infection appears to be limited by the amount of gene 4 protein, but too high a level of either 4A or 4B protein is inhibitory to growth.

Base Sequence↗

PubMed1992

Large scale purification and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation.

A rapid purification procedure produces milligram amounts of the T7 gene 4A' primase/helicase, 4B helicase, and the wild-type 4AB proteins expressed from the clones described in the accompanying paper (Rosenberg, A. H., Patel, S. S., Johnson, K. A., and Studier, F. W. (1992) J. Biol. Chem. 267, 15005-15012). Purified 4A' protein (in which the wild-type methionine at amino acid 64 has been replaced by leucine to eliminate the 4B initiation codon) appears to be equivalent to the wild-type 4A protein in primase, helicase, and NTPase activities. Gel filtration chromatography and polyacrylamide gel electrophoresis of native proteins indicate that the 4A' and 4B proteins form homodimers and heterodimers in solution. Heterodimer formation presumably accounts for an observed 3-fold increase in the primase activity of 4A' upon addition of 4B that lacks primase activity of its own. Steady-state k(cat) and Km values for hydrolysis of the nucleoside triphosphates ATP, dATP, dTTP, and dGTP were measured for 4A', 4B, 4A'B (1:1), and wild-type 4AB (1:2) proteins. The dependence of the dNTPase activities on the concentration was hyperbolic, suggesting single or noncooperative binding sites, whereas ATPase activity was sigmoidal, suggesting more than one ATP binding site. The k(cat)/Km ratios for hydrolysis of the dNTPs by the four protein preparations were within a factor of 6 of each other. The 1:1 mixture of 4A'B had the highest k(cat)/Km ratios, with a preference for dATP and dTTP.

Cell Line↗

PubMed1991

Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant.

The elementary steps of DNA polymerization catalyzed by T7 DNA polymerase have been resolved by transient-state analysis of single nucleotide incorporation, leading to the complete pathway: [formula: see text] where E, D, N, and P represent T7 DNA polymerase, DNA primer/template, deoxynucleoside triphosphate, and inorganic pyrophosphate, respectively. A DNA primer/template consisting of a synthetic 25/36-mer has been used as a substrate for correct nucleotide incorporation of dTTP in all the experiments. The rate constants and equilibrium constants of each step have been established by direct measurement of individual reactions and fit by computer simulation of the data to obtain a single set of rate constants accounting for all the data. Analysis of the single-turnover kinetics provided measurements of equilibrium dissociation constants for 25/36-mer, dTTP, and PPi equal to 18 nM (koff/kon), 18 microM (k-1/k1), and 2 mM (k5/k-5), respectively. The rate-limiting step during single-nucleotide incorporation has been identified as a conformational change, E.Dn.N----E'.Dn.N, which occurs at a rate of 300 s-1 (k2) upon binding of the correct dNTP. Accordingly, tighter binding of the transition states for the reaction resulting from the conformational change facilitates the phosphodiester bond formation. The chemical step itself was excluded as the rate-limiting step because of the small phosphothioate elemental effect. An observed rate constant of 70 s-1 for dTTP (alpha S) incorporation suggest that the chemical step (k3) occurs at a fast rate, greater than or equal to 9000 s-1. Following chemistry, the resulting ternary complex, E'.Dn+1.P, undergoes a second conformational change at a rate of 1200 s-1 (k4), leading to release of PPi and translocation of the DNA to continue subsequent cycles of polymerization. The rate constants of the reverse steps, 100 s-1 (k-2), greater than or equal to 18,000 s-1 (k-3) and 18 s-1 (k-4), were derived as fits to the data based upon simulation of single-turnover kinetics of pyrophosphorolysis including measurements of pyrophosphate exchange and the overall equilibrium constant of 1.0 x 10(4) for elongation of E.25/36-mer and analysis of the kinetics of the pulse-chase experiment. These studies provide the first complete and self-consistent thermodynamic descriptions of DNA polymerase and establish the basis for quantitative assessment of the reactions contributing to its extraordinary fidelity.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1991

An induced-fit kinetic mechanism for DNA replication fidelity: direct measurement by single-turnover kinetics.

An exonuclease-deficient mutant of T7 DNA polymerase was constructed and utilized in a series of kinetic studies on misincorporation and next correct dNTP incorporation. By using a synthetic oligonucleotide template-primer system for which the kinetic pathway for correct incorporation has been solved [Patel, S.S., Wong, I., & Johnson, K. A. (1991) Biochemistry (first of three papers in this issue)], the kinetic parameters for the incorporation of the incorrect triphosphates dATP, dCTP, and dGTP were determined, giving, respectively, kcat/Km values of 91, 23, and 4.3 M-1 s-1 and a discrimination in the polymerization step of 10(5)-10(6). The rates of misincorporation in all cases were linearly dependent on substrate concentration up to 4 mM, beyond which severe inhibition was observed. Competition of correct incorporation versus dCTP revealed an estimated Ki of approximately 6-8 mM, suggesting a corresponding kcat of 0.14s-1. Moderate elemental effects of 19-, 17-, and 34-fold reduction in rates were measured by substituting the alpha-thiotriphosphate analogues for dATP, dCTP, and dGTP, respectively, indicating that the chemistry step is partially rate-limiting. The absence of a burst of incorporation during the first turnover places the rate-limiting step at a triphosphate binding induced conformational change before chemistry. In contrast, the incorporation of the next correct triphosphate, dCTP, on a mismatched DNA substrate was saturable with a Km of 87 microM for dCTP, 4-fold higher than the Kd for the correct incorporation on duplex DNA, and a kcat of 0.025 s-1. A larger elemental effect of 60, however, suggests a rate-limiting chemistry step. The rate of pyrophosphorolysis on a mismatched 3'-end is undetectable, indicating that pyrophosphorolysis does not play a proofreading role in replication. These results show convincingly that the T7 DNA polymerase discriminates against the incorrect triphosphate by an induced-fit conformational change and that, following misincorporation, the enzyme then selects against the resultant mismatched DNA by a slow, rate-limiting chemistry step, thereby allowing sufficient time for the release of the mismatched DNA from the polymerase active site to be followed by exonucleolytic error correction.

DNA Replication↗

PubMed1991

Kinetic partitioning between the exonuclease and polymerase sites in DNA error correction.

We present a kinetic partitioning mechanism by which the highly efficient 3'----5' exonuclease activity of T7 DNA polymerase maximizes its contribution to replication fidelity with minimal excision of correctly base-paired DNA. The elementary rate constants for the proposed mechanism have been measured directly from single-turnover experiments by using rapid chemical quench-flow techniques. The exonuclease activity of T7 DNA polymerase toward single-stranded DNA is quite fast (kx greater than 700 s-1). This rapid exonuclease is restrained with double-stranded DNA by a kinetic partitioning mechanism that favors the binding of the DNA to the polymerase site to prevent the rapid degradation of matched DNA and yet allows selective removal of mismatched DNAs. Both matched and mismatched DNAs bind tightly to the polymerase site, with approximately equal affinities, Kdp = 20 and 10 nM, respectively. Selective removal of the mismatch is governed by the rate of transfer of the DNA from the polymerase to the exonuclease site (kp----x). The rapid excision of matched DNA is limited by a slow transfer rate (kp----x = 0.2 s-1) from the polymerase to the exonuclease site relative to the rate of polymerization [kp = 300 s-1; Patel et al. (1991) Biochemistry (first of three papers in this issue)]. Removal of mismatched DNA is facilitated by its faster transfer rate (kp----x = 2.3 s-1) to the exonuclease site relative to the slow rate of polymerization over a mismatch [kpi = 0.012 s-1; Wong et al. (1991) Biochemistry (second of three papers in this issue)].(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1991

Effects of thymidine and uridine on the phosphorylation of 3'-azido-3'-deoxythymidine (zidovudine) in human mononuclear cells.

The effects of thymidine and uridine on the phosphorylation of 3'-azido-3'-deoxythymidine (AZT) were studied in various human mononuclear cell preparations. Thymidine suppressed [3H]AZT phosphorylation in the same concentration range (20 to 100 microM) in which it antagonizes the anti-human immunodeficiency virus activity of AZT. Uridine, in turn, had no influence on AZT phosphorylation, just as it has no effect on the anti-human immunodeficiency virus activity of AZT. These findings are consistent with a close relationship between the inhibition of AZT phosphorylation and the influence of physiological nucleosides on the antiviral activity of AZT.

Bone Marrow↗

PubMed1991

Differential inhibition of 2'-deoxycytidine salvage as a possible mechanism for potentiation of the anti-human immunodeficiency virus activity of 2',3'-dideoxycytidine by dipyridamole.

Dipyridamole, a commonly used coronary vasodilator and antithrombotic drug, was recently shown to potentiate the activity of 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine against the human immunodeficiency virus type 1 (HIV-1) in human monocyte-macrophages in vitro. We report in the present paper that in uninfected monocyte-macrophages dipyridamole significantly inhibits cellular salvage of [3H]deoxycytidine, whereas it does not affect the salvage of [3H]dideoxycytidine. Similar differential inhibition by dipyridamole of the salvage of thymidine, as opposed to 3'-azido-3'-deoxythymidine, was reported previously (G. V. Betageri, J. Szebeni, K. Hung, S. S. Patel, L. M. Wahl, M. Corcoran, and J. N. Weinstein, Biochem. Pharmacol. 40:867-870, 1990). Taken together, these observations suggest that inhibition of the salvage of competing physiological nucleosides may explain or contribute to the potentiating effect of dipyridamole on these antiviral dideoxynucleoside drugs.

Antiviral Agents↗

PubMed1990

Effect of dipyridamole on transport and phosphorylation of thymidine and 3'-azido-3'-deoxythymidine in human monocyte/macrophages.

Dipyridamole (DPM), a commonly used coronary vasodilator and antithrombotic drug, was shown recently to potentiate the antiviral effect of 3'-azido-3'-deoxythymidine (AZT) in HIV-1 infected human monocyte-derived macrophages (M/M) in vitro. We report in the present study that in uninfected M/M, DPM markedly inhibited cellular uptake of [3H]thymidine (dThd) and its incorporation into the nucleotide pools, particularly the dThd-triphosphate pool. In contrast, DPM did not affect cellular uptake and phosphorylation of [3H]AZT. Since dThd counteracts the phosphorylation and antiviral action of AZT, these findings support the hypothesis that the potentiation of the anti-HIV effect of AZT is due, at least in part, to differential inhibition of nucleoside salvage.

Biological Transport↗

PubMed1990

Regulation of human B cell responsiveness by CD8+ T cells: differential effects of stimulation with monoclonal antibodies to CD3 and pokeweed mitogen.

Previous studies have shown that human CD8-positive T cells activated by immobilized mAb to the CD3 complex have the capacity to support the generation of Ig secreting cells (ISC). The experiments reported here were undertaken to examine the nature of CD8+ T cell helper function in greater detail. CD8+ T cells that had been treated with mitomycin C (CD8+ mito) and stimulated by immobilized mAb to CD3 (64.1) provided help for the generation of ISC from resting B cells. By contrast, CD8+ mito did not support the generation of ISC in cultures stimulated by pokeweed mitogen (PWM). This could not be explained by differences in the production of IL2, since PWM and anti-CD3 induced comparable amounts of IL2 from CD8+ mito. In anti-CD3-stimulated cultures, CD8+ mito supported the generation of larger numbers of ISC when B cells were also activated with Staphylococcus aureus (SA). By contrast, in PWM-stimulated cultures, CD8+ mito did not provide help for SA-activated B cells. Rather, PWM-stimulated CD8+ mito appeared to suppress the generation of ISC induced by PWM-activated CD4+ mito or by SA + IL2, whereas anti-CD3-stimulated CD8+ mito did not. Only control CD8+ T cells, which were able to proliferate, exerted suppressive effects in anti-CD3-stimulated cultures. Examination of the functional capacities of a battery of CD8+ T cell clones indicated that the same clonal population of CD8+ cells could provide help or suppress responses when stimulated with anti-CD3 or PWM, respectively. The functional activities of CD8+ clones differed from those of fresh CD8+ cells. Thus, anti-CD3-stimulated CD8+ clones provided help for B cells regardless of whether they were treated with mitomycin C. Moreover, PWM stimulated suppression by CD8+ clones was abrogated by treating the clones with radiation or mitomycin C. These results indicate that helper T cell function is not limited to the CD4+ T cell population, but that help can also be provided by appropriately stimulated CD8+ T cells. Taken together, these results indicate that CD8+ T cells are not limited in their capacity to regulate B cell responses, but rather can provide positive or negative influences depending on the nature of the activating stimulus.

Antibodies, Monoclonal↗

PubMed1989

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones.

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1989

Leukocyte function-associated antigen 1 is an activation molecule for human T cells.

The leukocyte function-associated antigen 1 (LFA-1) molecule is well established as a surface protein involved in cellular adhesion and interaction, but there has been little information about whether engagement of this molecule can also directly modify cellular activation. These studies demonstrate that crosslinking the LFA-1 molecule on human T cell clones transmits a unique signal to the cell. Crosslinking LFA-1 alone did not increase intracellular calcium ([ CA2+]i), nor did crosslinking LFA-1 activate the cells as measured by IL-2 production or [3H]thymidine incorporation. However, when CD3 and LFA-1 were crosslinked, a more prolonged calcium signal was observed than when CD3 alone was crosslinked. Moreover, IL-2 production and DNA synthesis were greatly augmented. These responses could be demonstrated when LFA-1 was crosslinked via either the alpha or the beta chain, and required surface expression of the LFA-1 molecule as no enhancement was observed in T cell clones from a child with leukocyte adhesion deficiency. The enhancement of cellular activation by LFA-1 did not require that it be directly crosslinked to the CD3 complex. Thus, crosslinking LFA-1 alone with isotype-specific secondary antibodies on cells also pretreated with an anti-CD3 mAb of a different Ig isotype stimulated the cells as effectively as crosslinking both surface antigens with GaMIg. Similarly, a delayed, but sustained increase in [Ca2+]i was elicited. This increase in [Ca2+]i and the enhanced functional responses required engagement of CD3 with an intact bivalent anti-CD3 mAb, as crosslinking LFA-1 on cells also reacted with Fab fragments of an anti-CD3 mAb did not increase [Ca2+]i, nor activate the cells. These data indicate that LFA-1 can convey activation signals to T cells. Synergism in signaling can be observed upon crosslinking of LFA-1 and independently crosslinking CD3. In the physiologic interaction between T cells and accessory cells, the interaction of LFA-1 with its ligand, intercellular adhesion molecule 1, may therefore not only facilitate cellular adhesion, but also may amplify T cell activation by delivering costimulatory signals.

Antibodies, Monoclonal↗

PubMed1989

Patterns of costimulation of T cell clones by cross-linking CD3, CD4/CD8, and class I MHC molecules.

The ability of mAb to class I MHC molecules, CD3, or CD4/CD8 to stimulate human T cell clones alone or in combination was examined. Cross-linking each of these surface Ag with appropriate mAb and goat anti-mouse Ig (GaMIg) resulted in a unique pattern of increase in intracellular free calcium ([Ca2+]i) and different degrees of functional activation. Cross-linking class I MHC molecules provided the most effective stimulus of IL-2 production and proliferation. Cross-linking more than one surface Ag induced a compound calcium signal with characteristics of each individual response. Cross-linking CD3 + HLA-A,B,C caused a rapid and prolonged increase in [Ca2+]i and synergistically increased IL-2 production and proliferation of all clones. Cross-linking CD3 + CD4/CD8 also generated a compound calcium signal and increased IL-2 production and DNA synthesis. Purposeful inclusion of CD3 was not required for costimulation as cross-linking HLA-A,B,C + CD4/CD8 also increased [Ca2+]i, IL-2 production, and proliferation. Cross-linking three surface Ag, CD3 + HLA-A,B,C + CD4/CD8, resulted in the greatest initial and sustained [Ca2+]i, IL-2 production, and DNA synthesis. Although there was a tendency for the various stimuli to increase both [Ca2+]i and functional responsiveness, neither the magnitude nor duration of the increased [Ca2+]i correlated with the amount of IL-2 produced or the ultimate proliferative response. To determine whether costimulation required that the various surface molecules were cross-linked together, experiments were carried out using isotype specific secondary antibodies. Augmentation of [Ca2+]i and costimulation of functional responses were noted when class I MHC molecules were cross-linked and CD3 was bound, but not cross-linked. Similarly, costimulation through CD3 and CD4/CD8 was observed when CD4/CD8 was cross-linked and the CD3 complex was engaged by an anti-CD3 mAb which was not further cross-linked. In contrast, costimulation by class I MHC molecules and CD4/CD8 was only observed when these molecules were cross-linked together. These data demonstrate that cross-linking class I MHC determinants or CD4/CD8 provides a direct signal to T cell clones that can be enhanced when CD3 is independently engaged. The results also indicate that T cell clones can be stimulated without engaging CD3 by the combination of signals delivered via class I MHC molecules and CD4/CD8, but only when these determinants were cross-linked together. These studies have demonstrated that these cell surface molecules differ in their capacity to deliver activation signals to T cell clones and also exhibit unique patterns of positive cooperativity in signaling potential.

Adjuvants, Immunologic↗

PubMed1989

Activation of human T cell clones and Jurkat cells by cross-linking class I MHC molecules.

Cross-linking class I MHC molecules on human T cell clones by reacting them with various mAb directed at either monomorphic or polymorphic determinants on class I MHC molecules followed by cross-linking with GaMIg stimulated a rise in intracellular free calcium concentration ([Ca2+]i), and induced proliferation and IL-2 production. T cell clones varied in the mean density of class I MHC molecules and the capacity to respond to mAb to class I MHC molecules. However, the functional responses of the clones did not correlate with class I MHC density or the CD4/CD8 phenotype. mAb to polymorphic class I MHC determinants were less able to induce an increase in [Ca2+]i and a functional response in the T cell clones. Additive stimulatory effects were noted when mAb against both HLA-A and HLA-B determinants were employed. Cross-linking class I MHC molecules on Jurkat cells induced a rise by [Ca2+]i and induced IL-2 production upon co-stimulation with PMA. Cross-linking class I MHC molecules on mutant Jurkat cells that expressed diminished levels of CD3 and were unable to produce IL-2 in response to anti-CD3 stimulation triggered both a rise in [Ca2+]i and IL-2 production with PMA co-stimulation. In contrast, cross-linking class I MHC molecules on mutant Jurkat cells that were CD3- stimulated neither a rise in [Ca2+]i nor IL-2 production. The combination of mAb to CD28 or ionomycin and PMA, however, was able to induce IL-2 production by CD3- Jurkat cells. The data demonstrate that cross-linking class I MHC molecules delivers a functionally important signal to T cell clones and Jurkat cells and indicate that class I MHC molecules may function to transduce activation signals to T cells. In addition, the data demonstrate that transmission of an activation signal via class I MHC molecules requires CD3 expression. The data, therefore, support a central role for CD3 in the transduction of activation signals to T cells via class I MHC molecules.

Antibodies, Monoclonal↗