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F Ivars

Publications and source records attributed to F Ivars.

At least 19 recordsLinked to original sources

C-Med 100, a hot water extract of Uncaria tomentosa, prolongs lymphocyte survival in vivo.

Water extracts of the bark of Uncaria tomentosa, a vine indigenous to South America, has been used for generations as an "immuno modulator". To understand the basis of this immuno modulatory effect we fed mice in their drinking water with C-Med 100, which is a commercially available water extract from Uncaria tomentosa. We found a dose-dependent increase in spleen cell numbers in the supplemented mice, but the proportions of B cells, T cells, NK cells, granulocytes, and memory lymphocytes were normal. However, there were no detectable changes of the lymphoid architecture of the spleen even after long-term treatment. Further, when C-Med 100 treatment was interrupted the cellularity returned to normal level within four weeks. The increased number of lymphocytes was most likely not due to increased production because C-Med 100 did not have any significant effect on precursor cells nor on the accumulation of recent thymic emigrants in the spleen. We conclude that accumulation is most likely due to prolonged cell survival, because adoptive transfer experiments demonstrated that C-Med 100 treatment significantly prolonged lymphocyte survival in peripheral lymphoid organs, without increasing their proliferation rate. Since the accumulation was reversible and without detectable pathological effects, these results suggest the use of C-Med 100 as a potential agent for clinically accelerating the recovery of patients from leukopenia.

Adjuvants, Immunologic↗

A pTalpha-negative subpopulation of CD25+ TN thymocytes revealed by a transgenic marker.

We have recently generated 5'lambda5-huTAC mice, which express the human CD25 (huTAC) gene under the control of the 5'-flanking region of the mouse lambda5-gene. The huTAC-transgene was expressed in pre-B cells but neither in mature B cells nor in T cells of these mice. In this report we demonstrate that the transgene is also transiently expressed by adult CD25+ CD3-CD4-CD8- (triple negative, TN) thymocytes and in fetal thymocytes. The huTAC+, in contrast to the huTAC- subpopulation of the CD44+CD25+ TN cells, was unexpectedly found not to express the pTalpha-gene. Still the huTAC+CD44+CD25+ TN cells reconstituted the development of both alphabeta and gammadelta lineage cells equally efficiently as the pTalpha-expressing huTAC- fraction, demonstrating that this pTalpha-negative subpopulation contained precursors for both T-cell lineages. Single cell reverse transcription-polymerase chain reaction (RT-PCR) experiments demonstrated that also in normal mice only a fraction of CD44+CD25+ and CD44-CD25+ TN cells expressed this gene. Taken together, these data indicate that huTAC transgene expression revealed a truly pTalpha-negative fraction of the CD44+CD25+ TN cells. The observation that not all precursors in the CD25+ TN population express the pTalpha-gene has important implications for the understanding of early T-cell development and T-cell lineage commitment.

Animals↗

Early TCR alpha beta expression promotes maturation of T cells expressing Fc epsilon RI gamma containing TCR/CD3 complexes.

In a previous study we presented data indicating that the expanded population of CD4(-)CD8(-) (DN) alphabeta T cells in TCRalpha-chain-transgenic mice was partially if not entirely derived from gammadelta T cell lineage cells. The development of both gammadelta T cells and DN alphabeta T cells is poorly understood; therefore, we thought it would be important to identify the immediate precursors of the transgene-induced DN alphabeta T cells. We have in this report studied the early T cell development in these mice and we show that the transgenic TCRalpha-chain is expressed by precursor thymocytes already at the CD3(-)CD4(-)CD8(-) (triple negative, TN) CD44(+)CD25(-) stage of development. Both by using purified precursor populations in reconstitution experiments and by analyzing fetal thymocyte development, we demonstrated that early TN precursors expressing endogenous TCRbeta-chains matured into DN alphabeta T cells at several stages of development. The genes encoding the gamma-chain of the high affinity receptor for IgE (FcepsilonRIgamma) and the CD3zeta protein were found to be reciprocally expressed in TN thymocytes such that during development the FcepsilonRIgamma expression decreased whereas CD3zeta expression increased. Furthermore, in a fraction of the transgene-induced DN alphabeta T cells the FcepsilonRIgamma protein colocalized with the TCR/CD3 complex. These data suggest that similarly to gammadelta T cells and NKT cells, precursors expressing the TCR early in the common alphabetagammadelta developmental pathway may use the FcepsilonRIgamma protein as a signaling component of the TCR/CD3 complex.

Animals↗

N-substituted benzamides inhibit nuclear factor-kappaB and nuclear factor of activated T cells activity while inducing activator protein 1 activity in T lymphocytes.

N-substituted benzamides are compounds that have recently been reported to inhibit nuclear factor-kappaB (NF-kappaB) activity and induce apoptosis in a pre-B cell line. In this study, we focused on the effects of N-substituted benzamides on transcriptional regulation in Jurkat T cells. We used a model system where the cells can be stimulated either through TCR/CD28 or by treatment of the cells with PMA and ionomycin to induce transcription factors typical for T lymphocyte activation. Treatment of the Jurkat cells with procainamide did not influence the transcription factor profile of stimulated cells, while treatment with a derivative having an acetyl group in position 4 of the aromatic ring inhibited NF-kappaB and nuclear factor of activated T cells (NFAT) activity. Declopramide, which contains a chloride in position 3 of the aromatic ring, was inactive in this system, whereas also the acetylated derivative of this compound inhibited NF-kappaB and NFAT activity. In contrast, the transcriptional activity and nuclear expression of activator protein 1 induced by TCR/CD28 stimulation or PMA and ionomycin treatment was enhanced by the acetylated variants of the N-substituted benzamides. Finally, we investigated the effect of N-substituted benzamides on intact promoters for two genes central in immune regulation; the CD40 ligand (CD40L) and IL-2 promoters. The transcriptional activity of the CD40L promoter as well as surface expression of the CD40L induced by signaling through TCR/CD28 was inhibited by addition of acetylated N-substituted benzamides, while the transcriptional activity of the IL-2 promoter was enhanced. Taken together, these data indicate that derivatives of N-substituted benzamides are potential drug candidates for quantitative as well as qualitative modulation of immune functions.

Acecainide↗

CD4+CD25+ regulatory T cells down-regulate co-stimulatory molecules on antigen-presenting cells.

CD4+CD25+ T cells have been shown to inhibit experimentally induced organ-specific autoimmune disease and depletion of these regulatory T cells from normal mice results in development of such conditions. Furthermore, CD4+CD25+ T cells suppress the IL-2 production and thereby the proliferation of polyclonally activated CD4+CD25- T cells in vitro. The suppression in vitro is independent of secreted factors but requires interactions between CD4+CD25- and CD4+CD25+ T cells and antigen-presenting cells (APC). We have now further investigated the function of CD4+CD25+ T cells in vitro and have focused on their interactions with APC. We found that CD4+CD25+ T cells down-regulated the expression of the co-stimulatory molecules CD80 and CD86 on dendritic cells. The steady-state level of CD80 mRNA was also decreased, while the steady-state level of CD86 mRNA was not, suggesting that distinct mechanisms regulate the expression of these molecules. The down-regulation occurred even in the presence of stimuli that would normally increase the expression of CD80 and CD86 molecules. Thus, down-regulation of co-stimulatory molecules may be an additional effector function of these regulatory T cells.

Animals↗

Characterization of subpopulations of T-cell receptor intermediate (TCRint) T cells.

CD1-autoreactive T cells of two types have been demonstrated among T cells expressing the T-cell receptor (TCR) alphabeta at intermediate levels (TCRint cells). One type constitutes a major fraction of the natural killer (NK)1.1+ TCRint population in C57BL/6 (B6) mice and carries a restricted TCR composed of an alpha-chain with an invariant Valpha14-J281 rearrangement, and a beta-chain using Vbeta8. 2, 7 or 2. The second type utilises a variety of TCR and was derived from CD4+ cells in mice lacking MHC class II. To increase our understanding of the two different CD1-reactive subsets, we have investigated and compared the populations of origin: NK1.1+ and NK1. 1- TCRint subsets from MHC class II-deficient mice and CD4+NK1.1+ T cells from B6 mice. The three TCRint populations shared a phenotype indicating previous activation, and contained low frequencies of cells expressing NK receptors of the Ly49 family. In contrast to control CD4+ cells, the three TCRint subsets produced high amounts of interleukin (IL)-4 and interferon (IFN)-gamma after activation. Importantly, no IL-10 could be detected in either TCRint population, implying a distinct function for these cells, different from those of conventional CD8+ and CD4+ cells, including the typical T-helper 2 (Th2) cell. Analysis of TCR expression indicated that the proportion of cells using the semi-invariant Valpha14/Vbeta8.2-type TCR was lower in NK1.1+ cells from MHC class II-negative mice than in CD4+NK1.1+ B6 cells. Further, usage of the Valpha14-J281 rearrangement was also demonstrated among NK1.1- TCRint cells.

Animals↗

A TCR alpha chain transgene induces maturation of CD4- CD8- alpha beta+ T cells from gamma delta T cell precursors.

The proportion of CD4- CD8- double-negative (DN) alpha beta T cells is increased both in the thymus and in peripheral lymphoid organs of TCR alpha chain-transgenic mice. In this report we have characterized this T cell population to elucidate its relationship to alpha beta and gamma delta T cells. We show that the transgenic DN cells are phenotypically similar to gamma delta T cells but distinct from DN NK T cells. The precursors of DN cells have neither rearranged endogenous TCR alpha genes nor been negatively selected by the MIsa antigen, suggesting that they originate from a differentiation stage before the onset of TCR alpha chain rearrangements and CD4/CD8 gene expression. Neither in-frame V delta D delta J delta nor V gamma J gamma rearrangements are over-represented in this population. However, since peripheral gamma delta T cells with functional TCR beta gene rearrangements have been depleted in the transgenics, we propose that the transgenic DN population, at least partially, originates from the precursors of those cells. The present data lend support to the view that maturation signals to gamma delta lineage-committed precursors can be delivered via TCR alpha beta heterodimers.

Animals↗

Naturally activated CD4+ T cells are highly enriched for cytokine-producing cells.

Most T cells in a normal non-immunized individual are in a resting state. However, a small proportion of splenic T cells are large activated cells both in specific pathogen-free and antigen-free mice. To further elucidate the effector functions associated with these "naturally" activated CD4+ T cells, we have characterized the expression of various membrane markers, cytokine production and T helper activity by these cells. We show that naturally activated CD4+ T cells express activation markers and contain tenfold higher proportions of cells producing IL-4, IL-10 and IFN-gamma as compared to small CD4+ T cells. Despite the high proportion of IFN-gamma producers, naturally activated CD4+ T cells still induce B cell proliferation and differentiation. These results are discussed in the context of normal physiological autoreactivity.

Animals↗

Gammadelta T-cell precursor-derived CD4- CD8- alphabeta T cells retain gammadelta cell function.

We have previously shown that some of the DN alphabeta+ T cells arising in TcR alpha-chain transgenic mice are of gammadelta T cell origin, based on phenotypic data and on their status of TcR gene rearrangements. In the present report we investigated the impact of alphabeta TcR expression on the functional programme of the mature gammadelta precursor-derived DN alphabeta+ T cells. Our results demonstrate that both their proliferative capacity and their cytokine production profile are similar to that of gammadelta T cells. Furthermore, both transgenic DN alphabeta+ T cells and DN gammadelta+ T cells up-regulate CD8alpha expression after activation, but, in contrast to CD4+ alphabeta T cells, are unable to induce proliferation of naive B cells. Thus, our results suggest that the effector functions of mature T cells are determined independently of the TcR isotype, probably at an early stage of differentiation, and thereby have important implications for current models of T-cell lineage commitment.

Animals↗

T cell subset-specific expression of antigen receptor beta chains in alpha chain-transgenic mice.

A large number of V beta 8 gene-encoded cDNA were analyzed from peripheral CD4+ and CD8+ T cell subsets of T cell receptor (TcR) alpha chain-transgenic mice. This analysis demonstrates that a limited repertoire of TcR beta chains are co-expressed with the transgenic alpha chain. Most importantly, certain V beta 8-J beta combinations were found exclusively in one of the subsets and, in some cases, subset-specific differences were localized to the VDJ junctional region of the beta chain genes. In contrast, CD4-CD8- transgenic T cells, as well as CD4+ and CD8+ T cells from normal littermate controls, were found to express diverse beta chain repertoires. The present study suggests that beta chains with distinct structural characteristics are expressed in the CD4+ and CD8+ subsets, respectively. Moreover, the data suggest that the same structural constraints do not apply to the population of CD4-CD8- transgenic T cells.

Amino Acid Sequence↗

Signal transduction through the T cell receptor-CD3 complex. Evidence for heterogeneity in receptor coupling.

T cell receptor-CD3 complex (TCR-CD3)-mediated signal transduction was analyzed in HPB-ALL and Jurkat T cell lines. Both cell lines express high levels of TCR-CD3 complex on the cell surface, but provide different model systems for TCR-CD3 signaling in T cells. Jurkat responds with both inositol phosphate generation and intracellular Ca2+ mobilization after triggering of TCR-CD3, whereas TCR-CD3 triggering of HPB-ALL induces Ca2+ mobilization without detectable inositol phosphate generation. By employing a permeabilized cell system, we show that the HPB-ALL line expressed normal levels of Ca2(+)-induced phospholipase C activity. However, the TCR-CD3 on this cell line seems to be uncoupled from phospholipase C activation. In agreement with this result we also show, by analysis of protein kinase C-dependent phosphorylation of three distinct substrates, that TCR-CD3 in HPB-ALL is apparently uncoupled from protein kinase C activation. These findings may have implications for understanding signal-transducing pathways in T cells at various stages of differentiation.

Antigens, CD↗

Expression of T-cell receptor alpha-chain genes in transgenic mice.

To examine the influences responsible for shaping the T-cell repertoire in vivo, we have introduced T-cell receptors of defined specificity into mice. In this report, we analyze transgenic mice carrying a T-cell receptor alpha-chain gene from a pigeon cytochrome c-reactive T-cell line. A variant of this construct, which has the immunoglobulin heavy-chain enhancer inserted into the JC intron, was also introduced into mice. Addition of the enhancer increased the steady-state level of transgene-encoded mRNA three- to fivefold in cultured T cells, leading to a two- to threefold increase in surface expression. In vivo, the difference between these two constructs was even more significant, increasing the number of transgene-positive cells from approximately 5 to 70% and the T-cell receptor surface density two- to threefold. Surprisingly, while surface expression of either type of transgene was limited to T cells, we found little tissue specificity with respect to transcription. In T cells expressing the alpha chain from the enhancer-containing construct, immunoprecipitation with a 2B4 alpha-specific monoclonal antibody revealed the expected disulfide-linked dimer. Costaining of these T cells with the 2B4 alpha-specific monoclonal antibody versus anti-CD3 indicated that expression of the transgene-encoded alpha chain precludes expression of endogenous alpha chains on the majority of cells; in contrast, 2B4 alpha-chain expression from the construct lacking the enhancer is inefficient at suppressing endogenous alpha-chain expression. In mice of the enhancer lineage, Southern blot analysis indicated suppression of endogenous alpha-chain rearrangements in T-cell populations, consistent with the observed allelic exclusion at the cellular level. Interestingly, newborn, but not adult, mice of this lineage also showed an increase in retention of unrearranged delta-chain loci in thymocyte DNA, presumably resulting from the suppression of alpha-chain rearrangements. This observation indicates that at least a fraction of alpha:beta-positive T cells have never attempted to produce functional delta rearrangements, thus suggesting that alpha:beta and gamma:delta T cells may be derived from different T-cell compartments (at least during the early phases of T-cell differentiation).

Alleles↗

The immune response to bacterial dextrans. V. A "dominant" idiotype in IgCHb mice.

A battery of syngeneic monoclonal anti-idiotypic antibodies was prepared against a monoclonal C57BL/6 anti-Dextran B512 antibody (17-9). Two such anti-idiotypes were shown to bind to sites on the 17-9 molecule which are related to the Dex-binding site and were used to characterize the anti-Dex antibody response in a number of inbred mouse strains. The results show that the 17-9 idiotype is recurrently expressed by all mice carrying the IgCHb haplotype, regardless of H-2 or background genes, and that this idiotype accounts for roughly one-half of the primary, specific response to Dex B512 in C57BL/6 mice. Backcross analysis confirmed the allotype-linkage of idiotype expression in the antibody response. Mice carrying other allotypes, however, had detectable levels of the 17-9 idiotype in normal sera, which was not associated with anti-Dex antibody activity and was not raised by specific immunization. Together with previous observations, these results characterize a second "recurrent" idiotype in the anti-alpha,1-6 response of IgCHb mice, both of which are expressed in the normal serum of all mouse strains tested.

Animals↗

The immune response to bacterial dextrans. III. Ontogenic development and strain distribution of specific clonal precursors.

The frequencies of B512 dextran (Dex)-specific B cell precursors were determined by limiting dilution analysis in a number of mouse strains originally described as "high responder", "low responder" and "nonresponder" to this antigen. No significant difference in the frequencies of Dex-specific precursors was found in C57BL/6, B10.BR, C3H/Tif, BALB/c and A/Sn adult mice. Together with the large intra-strain variability in the magnitude of anti-Dex PFC responses in vivo, these results established that differential reactivity in vivo cannot be ascribed to genetically controlled absence or wide variation in the frequency of Dex-specific immunocompetent precursors. A similar analysis of the Dex-specific precursor frequency was carried out in C57BL/6 mice between 1 week and 3 months of age. While no Dex-specific antibody response was detected in vivo before the age of 3 weeks, clonal precursor analysis revealed that the appearance of these specificities parallels the development of competent (IgM-producing) B lymphocyte clonal precursors, such that no significant difference in absolute frequencies of Dex-specific precursors could be observed among these age groups. This is interpreted to suggest that the late development of the Dex-specific antibody responses is regulatory rather than due to late rearrangement and activation of the appropriate V genes and a sequential expression of antibody specificities in ontogenic development.

Age Factors↗

Absence of immunoglobulin heavy chain expression results in altered kappa/lambda light chain ratios.

A few hundred hybridoma cell lines derived from spleen cells of normal, nonimmunized, 6-day-old BALB/c or BALB.B10 mice were screened for H- and L-chain production. Roughly half of these hybridomas produced no Ig chain into the culture supernatants. Of the Ig-secretors, 80% produced IgM molecules carrying either kappa or lambda chains in a ratio of 9:1. A surprisingly high frequency of clones (roughly 20%) were found to produce only light chains and in this group, the kappa/lambda ratio was altered to around 1. The indications from the primary screening were confirmed by isolating a large number of these hybrid cell lines and extensive testing of both culture supernatants and ascitic fluids in solid phase RIA and ELISA assays. In some cases, the absence or presence of heavy chain synthesis and the isotype of the light chain was also confirmed by biosynthetic labeling of the hybridoma clones, specific immuno-precipitation of cell lysates or supernatants, followed by analysis in SDS polyacrylamide gels. We conclude from these results that the absence of heavy chain expression is accompanied by frequencies of lambda chain expression, which are much higher than those observed in normal immunoglobulin producing cells, and equal to those of kappa chains. This conclusion can be interpreted as to suggest that the low frequency of lambda producing cells in mouse is in part determined by restrictions in the heavy-light chain interactions at the protein level.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗

Reactions among IgM antibodies derived from normal, neonatal mice.

Attempting to obtain a representative sample of the "natural antibody" repertoire in the developing immune system, we have derived IgM-secreting hybridomas from 4 normal untreated BALB/c mice of the same litter on day 6 after birth. Partially purified IgM preparations obtained in the supernatants of 70 such clones were each screened in binding assays for reactivity with a panel of 9 IgM antibodies, randomly selected from the same collection. Five of these 9 IgM antibodies were found to react with a considerable number of other IgM in the collection, while the other 4 showed only sporadic reactivity. On the other hand, more than half of the 70 antibodies were found to bind specifically to at least one of these five. With a few exceptions, these reactions showed quantitative levels ranging from 5 to 20% of those observed between either of the two interacting IgM and monoclonal rat anti-mu antibodies. The selectivity of these reactions indicated V-region specificity, which was confirmed by analyzing in some detail the reaction between 2 IgM antibodies isolated from the same mouse.

Animals↗

Idiotypic determinants of natural IgM antibodies that resemble self Ia antigens.

A collection of immunoglobulin-secreting B-cell hybridomas was derived from normal neonatal BALB/c spleen and searched for reactivity against a panel of monoclonal anti-H-2 antibodies. We describe here one IgM antibody which was found to react with the monoclonal anti-Ia.7 antibody 14-4-4S. The characterization of this clone (BA.N 4:4.57) revealed its anti-trinitrophenyl specificity and demonstrated specific binding to five different monoclonal anti-Ia.7 antibodies but not to other anti-H-2 antibodies. The variable region specificity of these interactions was shown by the use of pepsin Fab fragments of the IgM antibody. Anti-Ia.7 antibodies were shown to specifically inhibit plaque formation by the hybridoma cells, and dinitrophenylglycine was shown to inhibit the reaction between the IgM antibody and anti-Ia.7 molecules. We interpret these results as to indicate that BA.N 4:4.57 expresses an idiotope or idiotopes which mimic Ia.7 determinants. This idiotypic family is naturally expressed in both newborn and adult BALB/c mice, as shown by the presence in normal serum of IgM molecules that specifically react with the F(ab')2 fragment of the 14-4-4S antibody. We speculate on the importance of idiotypic mimicry with major histocompatibility complex determinants, for both the selection of natural antibody repertoires and the evolution of antibody genes.

Animals↗