Biomedical subjects

H T Petrie

Publications and source records attributed to H T Petrie.

At least 19 recordsLinked to original sources

PubMed1996

TCR gene recombination and alpha beta-gamma delta lineage divergence: productive TCR-beta rearrangement is neither exclusive nor preclusive of gamma delta cell development.

Two types of T lymphocytes can be generated intrathymically, distinguishable by either TCR-gamma delta or -alpha beta surface expression. Regulation of the intrathymic divergence of these cells is unresolved, at least in part because thymically derived gamma delta cells have rarely been studied. We used quantitative Southern blotting together with PCR-based cloning/sequencing and restriction fragment length polymorphism to analyze TCR-alpha and -beta gene recombination in thymically derived gamma delta cells. We found that TCR-beta gene recombination is a frequent occurrence in thymic gamma delta cells. Furthermore, not only do complete (V-D-J) TCR-beta gene rearrangements occur in thymic gamma delta cells, but the frequency of in-frame rearrangements is greater than would be predicted based upon random occurrence. In contrast, we show that thymically derived gamma delta cells do not make detectable rearrangements of the TCR-alpha locus. These studies clearly demarcate a point for alpha beta vs gamma delta commitment in the thymus, after TCR-beta but before TCR-alpha gene recombination. Further, while our data support gamma delta lineage commitment as a consequence of successful TCR-gamma and -delta gene rearrangement, we do not find support for a competitive model of lineage commitment, since productive TCR-beta gene rearrangement does not necessarily relegate cells to the alpha beta lineage.

Animals↗

PubMed1996

Regulation of cell division cycle progression by bcl-2 expression: a potential mechanism for inhibition of programmed cell death.

Expression of the bcl-2 gene has been shown to effectively confer resistance to programmed cell death under a variety of circumstances. However, despite a wealth of literature describing this phenomenon, very little is known about the mechanism of resistance. In the experiments described here, we show that bcl-2 gene expression can result in an inhibition of cell division cycle progression. These findings are based upon the analysis of cell cycle distribution, cell cycle kinetics, and relative phosphorylation of the retinoblastoma tumor suppressor protein, using primary tissues in vivo, ex vivo, and in vitro, as well as continuous cell lines. The effects of bcl-2 expression on cell cycle progression appear to be focused at the G1 to S phase transition, which is a critical control point in the decision between continued cell cycle progression or the induction programmed cell death. In all systems tested, bcl-2 expression resulted in a substantial 30-60% increase in the length of G1 phase; such an increase is very substantial in the context of other regulators of cell cycle progression. Based upon our findings, and the related findings of others, we propose a mechanism by which bcl-2 expression might exert its well known inhibition of programmed cell death by regulating the kinetics of cell cycle progression at a critical control point.

Animals↗

PubMed1995

T cell receptor gene recombination patterns and mechanisms: cell death, rescue, and T cell production.

The antigen-specific receptors of T and B lymphocytes are generated by somatic recombination between noncontiguous gene segments encoding the variable portions of these molecules. The semirandom nature of this process, while desirable for the generation of diversity, has been thought to exact a high price in terms of sterile (out-of-frame) products. Historically, the majority of T lymphocytes generated in mammals were thought to be useless, either because they generated such sterile rearrangements or because the receptors generated did not appropriately recognize self-molecules (i.e., positive and negative selection). In the studies described here, we characterize the onset of T cell receptor (TCR) alpha and beta chain gene rearrangements and quantitate their progression throughout T cell development. The results show that T cell production efficiency is enhanced through (a) rearrangement of TCR-beta chain genes early during T cell development, with selective expansion of those cells possessing in-frame rearrangements; (b) deletion of sterile rearrangements at the TCR-alpha chain locus through ordered (proximal to distal) sequential recombination; and (c) modification of nonselectable alpha/beta heterodimer specificities through generation and expression of new TCR-alpha chains. In addition, we demonstrate strict correlations between successful TCR-beta gene rearrangement, the onset of TCR-alpha gene rearrangement, rapid cell division, and programmed cell death, which together serve to maintain cell turnover and homeostasis during T cell development.

Animals↗

PubMed1995

In-frame TCR delta gene rearrangements play a critical role in the alpha beta/gamma delta T cell lineage decision.

Using a quantitative multiprobe Southern blot analysis, we demonstrate the surprising result that a significant proportion of alpha beta T cells and thymocytes retain T cell receptor delta locus sequences. A substantial portion of the retained delta locus is in a fully V-to-D-to-J rearranged configuration and 20% of these delta rearrangements are functional, significantly less than the 33% predicted for random gene rearrangements. Our observations are in conflict with the idea that alpha beta and gamma delta T cells derive from distinct precursors and suggest that commitment of a common precursor to the gamma delta lineage depends upon expression of a gamma delta T cell receptor. We propose that the intrathymic T cell lineage decision is determined by a competition between the production of functional gamma delta and beta-pre-T cell receptor complexes.

Animals↗

PubMed1994

TCR selection and allelic exclusion in RAG transgenic mice that exhibit abnormal T cell localization in lymph nodes and lymphatics.

RAG-1 and RAG-2 are developmentally regulated genes that are essential for V(D)J recombination and lymphocyte development. Expression of RAG-1 and RAG-2 by thymocytes is normally limited to cells that have not completed selection. We have previously documented that persistent expression of the recombinase activating genes (RAG) in transgenic mice results in aberrant thymic development, altered lymphatic microanatomy, and a profound immunodeficiency. Here we further document the pathologic changes found in TG.RAG-1,2 mice and examine the role of TCR recombination and positive and negative thymic selection, as well as allelic exclusion, in the etiology of the phenotype. We find that neither selection nor TCR allelic exclusion can be overcome by transgenic expression RAG-1 and RAG-2 under the control of an lck promoter.

Alleles↗

PubMed1994

T cell receptor beta chain gene rearrangement and selection during thymocyte development in adult mice.

The identification is made in normal mice of the stages in T cell development at which the rearranged beta chain of the T cell receptor (TCR) is utilized to promote T cell maturation, independent of the TCR alpha chain. In addition, evidence is provided that utilization of beta chains in T cell progenitors does not preclude differentiation to TCR gamma delta + T cells. This is consistent with the view that an initial consequence of beta chain expression by early thymocytes is clonal expansion, increasing the size of the pool of useful precursors. This allows the proposal to be made that allelic exclusion may be a byproduct of cell cycle regulation during early thymocyte differentiation, which may in turn explain why the efficiency of allelic exclusion varies at different TCR or immunoglobulin loci.

Animals↗

PubMed1993

Multiple rearrangements in T cell receptor alpha chain genes maximize the production of useful thymocytes.

Peripheral T lymphocytes each express surface T cell receptor (TCR) alpha and beta chains of a single specificity. These are produced after random somatic rearrangements in TCR alpha and beta germline genes. Published model systems using mice expressing TCR alpha and/or beta chain transgenes have shown that allelic exclusion occurs conventionally for TCR-beta. TCR alpha chain expression, however, appears to be less strictly regulated, as endogenous TCR alpha chains are often found in association with transgenic TCR beta chains in TCR alpha/beta transgenic mice. This finding, coupled with the unique structure of the TCR alpha locus, has led to the suggestion that unlike TCR beta and immunoglobulin heavy chain genes, TCR alpha genes may make multiple rearrangements on each chromosome. In the current study, we demonstrate that the majority of TCR-, noncycling thymocytes spontaneously acquire surface expression of CD3/TCR. Further, we show that cultured immature thymocytes originally expressing specific TCR alpha and beta chains may lose surface expression of the original TCR alpha, but not beta chains. These data provide evidence that not only must multiple rearrangements occur, but that TCR alpha gene rearrangement continues even after surface expression of a TCR alpha/beta heterodimer, apparently until the recombination process is halted by positive selection, or the cell dies. Sequential rearrangement of TCR alpha chain genes facilitates enhanced production of useful thymocytes, by increasing the frequency of production of both in-frame rearrangements and positively selectable TCR alpha/beta heterodimers.

Animals↗

PubMed1993

CD4+8- and CD4-8+ mature thymocytes require different post-selection processing for final development.

Two primary types of TCR-alpha/beta+ T cells are found in the peripheral lymphoid system; CD4+8- T cells, with MHC-class II restricted TCR, and CD4-8+ T cells, which are MHC-class I restricted. Both lineages develop in the thymus from a series of common precursors. However, the precise stage at which they diverge, and the combination of factors that regulates such divergence, are not well defined. The up-regulation of CD3/TCR to high mature levels is thought to be an early event associated with positive selection for self-MHC recognition. Using purified cells from bcl-2 transgenic mice in order to overcome the limitations imposed by cell death on normal thymocytes, we find that a minor subset of CD4+8+ thymocytes expressing high levels of CD3/TCR gives rise to both CD4+8- and CD4-8+ mature cells upon intrathymic transplantation, but only to CD4-8+ in culture. Thus, in addition to demonstrating the dual lineage potential of this subset, these findings show that additional post-selection processing events are required for the production of mature thymocytes, and that CD4+8- and CD4-8+ subsets differ in the types of processing required.

Animals↗

PubMed1993

A novel population of natural killer progenitor cells isolated from human umbilical cord blood.

In this report, we describe the isolation of a unique subpopulation of CD7+ cells from human fetal blood. Umbilical cord blood was first immuno-rosette-depleted using T cell, B cell, granulocyte, and macrophage markers to isolate a Lin- population. The Lin- cells were further characterized by cell sorting. As expected, the CD34+Lin- population (30%) was homogeneous and highly enriched for hemopoietic progenitors. Somewhat surprisingly, the CD34-Lin- population was also shown to be relatively homogeneous, with over 95% of cells expressing CD7. This CD34-Lin-CD7+ population was shown to be negative for all other T cell markers tested (i.e., CD7+1-2-3-4-8-). However, approximately 30% of these cells were positive for the NK cell surface markers CD16 and CD56 (CD7+NK+). Both CD7+NK+ and CD7+NK- populations proliferated in response to stimulation in vitro with IL-2/PHA/PHA-conditioned medium. After such treatment, approximately 40% of the CD7+NK- acquired CD56 and 20% CD16, whereas about 20% of the CD7+NK+ population became CD2+. The significance of the 60% of CD7+NK- cells that did not acquire other markers remains to be determined. In addition, although neither population was cytotoxic when first isolated, both populations acquired the ability to lyse the NK target cell line K562 while cultured under these conditions. These data suggest that these two populations may represent a developmental sequence among NK cell precursors in human umbilical cord blood. Additional analysis of such precursors may be useful in understanding the ontogeny of NK cells in vivo.

Antigens, CD↗

PubMed1992

Commitment to the T cell receptor-alpha beta or -gamma delta lineages can occur just prior to the onset of CD4 and CD8 expression among immature thymocytes.

Two types of T lymphocytes, distinguishable by their surface expression of either the gamma delta or the alpha beta T cell receptor (TcR) for antigen, populate the periphery in the adult. In addition, immature precursors of both T cell types can be found in the thymus. While it is generally accepted that these two cell types represent distinct lineages, it is not known at which developmental stage these lineages diverge. The most mature thymocyte precursor population not yet expressing T lineage-specific surface markers (i.e. CD3, CD4, and CD8) is known to be capable of generating TcR-alpha beta T cells, and has been thought to be preprogrammed into the TcR-alpha beta lineage at an earlier developmental stage. We now show that this late-stage precursor is capable of giving rise to cells of both the TcR-alpha beta and -gamma delta lineages, both in vitro after intrathymic transplantation, and in vitro in simple culture medium or medium with cytokines. Thus it appears that the divergence of TcR-alpha beta and -gamma delta cells can occur at a relatively late stage of intrathymic development, just prior to the onset of CD4 and CD8 expression in most cells.

Animals↗

PubMed1991

Characterization of immature CD4+CD8-CD3- thymocytes.

Previously we have described (Hugo, P. et al., Int. Immunol. 1990. 2: 209) an immature CD4+CD8-CD3- thymocyte subset which is thought to be the counterpart of the CD4-CD8+CD3- subset. In this study we show that the ontogeny of these two subsets is parallel in fetal thymic organ culture. Extensive phenotypic characterization of CD4+CD8-CD3- cells reveals that they closely resemble CD4-CD8+CD3- thymocytes being: HSAhigh, Thy-1high, interleukin 2 receptor alpha chain negative, CD44-, H-2K+/-, CD5low, MEL-14low/intermediate, CD2+, LFA-1+ and MTS 35+. Finally, we show that the proportion of CD4+CD8-CD3- thymocytes is highly variable between mouse strains.

Animals↗

PubMed1991

Timing of deletion of autoreactive V beta 6+ cells and down-modulation of either CD4 or CD8 on phenotypically distinct CD4+8+ subsets of thymocytes expressing intermediate or high levels of T cell receptor.

In this paper we describe a differentiation sequence amongst adult murine thymocytes which goes from CD4+8+3lo(low) to CD4+8+3int(intermediate) to CD4+8+3hi(high) and then to mature single positive CD3hi thymocytes. Phenotypic characterization of CD4+8+3int/hi cells for a number of other surface markers is consistent with them being in transition from CD4+8+3lo phenotype to mature phenotype. The same observation was made for sensitivity towards ionomycin-mediated apoptosis. In the thymus of Mls-1a mice, where autoreactive TCR-V beta 6+ cells are negatively selected, deletion of TCR-V beta 6+ cells was first detected in the CD4+8+3int subset, and was complete by the CD4+8+3hi stage, suggesting that up-regulation of the TCR/CD3 complex is required for deletion of Mls-1a autoreactive thymocytes. No sign of apoptosis was detected among any fresh thymocyte subsets suggesting that apoptotic cells are rapidly cleared from the thymus. The CD4+8+3int/CD4+8+3hi cells are therefore populations in transit from the typical cortical thymocytes to the mature T-cells.

Animals↗

PubMed1990

Lineage relationships and developmental kinetics of immature thymocytes: CD3, CD4, and CD8 acquisition in vivo and in vitro.

T lymphocytes develop in the thymus from immunologically naive bone marrow precursors. Based on T cell receptor rearrangement and transcription, and thymic reconstitution potential, we have deduced a developmental sequence among immature thymocytes, before the acquisition of the lineage markers CD3, CD4, and CD8. In the current study, we have followed the ontogenic progression of the latter stages in this sequence, using two different systems: (a) in vivo, by direct injection into the thymus of nonirradiated, congenic recipients; and (b) in vitro, using culture medium without mitogens or cytokines. In vivo, the less mature Pgp-1- interleukin 2 receptor alpha-positive (IL-2R alpha+) CD3-4-8- subset (also heat-stable antigen high) requires 3 d before becoming predominantly IL-2R alpha- CD3lo4+ 8+ typical cortical-type cells, and at least 5 d before the appearance of any mature single-positive cells (CD3hi4+ 8- or CD3hi4-8+). However, these Pgp-1- IL-2R alpha+ precursors do not differentiate further in unstimulated culture. The more mature Pgp-1- IL-2R alpha- CD3-4-8- subset becomes primarily CD3lo4+ 8+ within 1 d after transplantation, and some mature single-positive progeny are evident by day 3. By 5 d, most of these Pgp-1-IL-2R alpha- precursor cells have become CD3hi, and have lost or are downregulating either CD4 or CD8. In culture, these Pgp-1- IL-2R alpha- cells also acquire high levels of CD4 and CD8 within 1 d, and low levels of CD3 by 2 d. However, they do not progress further to mature single positives in vitro, and most of them die by day 3. These experiments directly confirm our previously proposed developmental sequence, and demonstrate the kinetics of T lymphocyte production in a low-stress, steady-state environment.

Animals↗

PubMed1990

Development of immature thymocytes: initiation of CD3, CD4, and CD8 acquisition parallels down-regulation of the interleukin 2 receptor alpha chain.

We have previously identified a developmental sequence among immature thymocytes, prior to their expression of the lineage markers CD3, CD4, and CD8. This sequence is marked by transient expression of the interleukin 2 receptor alpha chain (IL 2R alpha). The most mature cells in this sequence (surface phenotype heat-stable antigen (HSA)++ Pgp-1- IL 2R alpha-) are the immediate precursors to CD4+CD8+ small cortical thymocytes, and have by definition been considered to be CD4-CD8-. We now show that these cells display low levels of surface CD4 and CD8, but not CD3. This low-level expression begins to appear immediately after the loss of IL 2R alpha expression. Northern blot analysis for mRNA expression confirms that these IL 2R alpha- cells are transcribing CD4 and CD8 mRNA, in contrast to their immediate (IL 2R alpha+) precursor. Upon unstimulated culture, these IL 2R alpha- cells gradually acquire high levels of CD4 and CD8, as well as low levels of CD3, whereas IL 2R alpha+ cells do not. These findings suggest that the IL 2R alpha+ subset is the end of the true CD3-CD4-CD8- phase, and that the intracellular signals for CD3, CD4, and CD8 acquisition occur simultaneously with, or immediately prior to, the signal for down-regulation of IL 2R alpha.

Animals↗

PubMed1989

Selenium and the immune response: 1. Modulation of alloreactive human lymphocyte functions in vitro.

A role for the dietary trace mineral element selenium in the reduction of cancer incidence has been documented in numerous epidemiological and experimental studies. The precise mechanism of this antitumor effect is not well understood, but published data suggest that both inhibition of tumor cell growth and enhancement of host immunity are likely to be involved. In this study we report that selenium at physiologic concentrations can inhibit human lymphocyte proliferation in response to irradiated tumor cells in mixed lymphocyte/tumor cell cultures (MLTC). In addition, we demonstrate that the various lymphocyte functional activities generated in these cultures exhibit different levels of sensitivity to the effects of selenium. The generation of suppressor-cell activity in MLTC was strongly inhibited by the presence of physiologic levels of selenium, while the development of cytotoxic T-lymphocyte activity in identical cultures was not affected by selenium. Production of interleukin-2 in these cultures showed an intermediate sensitivity to the effects of selenium. Thus, selenium appears to be capable of selectively regulating the generation of functional lymphocyte subsets in vitro. Such selective regulation could explain the published effects of selenium on immunity and would be consistent with a role for immunity in the observed reduction of cancer incidence associated with elevated selenium intake.

Humans↗

PubMed1989

Selenium and the immune response: 2. Enhancement of murine cytotoxic T-lymphocyte and natural killer cell cytotoxicity in vivo.

An inverse correlation between cancer incidence and dietary intake of the trace mineral element selenium has been well established in epidemiological and experimental studies. The mechanisms for this chemoprotective effect are unresolved. Much attention has been focused on the antiproliferative effects of selenium on various normal and neoplastic cell types. However, dietary selenium supplementation can also enhance the expression of various humoral and cellular immune responses. In examining the effects of dietary selenium on cell-mediated immunity in mice, we observed that selenium supplementation caused the enhanced expression of spontaneous natural killer (NK) cytotoxicity in spleen cells and of specific cytotoxic T-lymphocyte (CTL) cytotoxicity in peritoneal exudate cells (PEC). NK activity of spleen-cell suspensions from selenium-supplemented mice increased an average of 70% over that of the control group (basal diet). Cytotoxic activity of PEC from mice injected with tumors intraperitoneally peaked earlier in selenium-supplemented animals, and the appearance of cells staining positively for Thy 1.2 surface antigen in selenium-supplemented animals also preceded the values observed in control animals. We propose here that enhancement of in vivo cytotoxic mechanisms, is likely to act synergistically with tumor growth inhibition in the reduction of tumor incidence associated with selenium intake.

Animals↗

PubMed1989

The acquisition of CD4 and CD8 during the differentiation of early thymocytes in short-term culture.

Subpopulations of thymocytes known to represent early stages of T cell development were isolated from the adult mouse thymus, and their ability to differentiate during short periods of culture was assessed by their acquisition of surface CD4 and CD8. Virtually all cells of the most mature of the CD4-CD8- thymocyte subpopulations (other surface markers CD3- HSA++ IL-2R-Pgp-1-) and of the immature CD4-CD8+ thymocyte subpopulation (other surface markers also CD3- HSA++ IL-2R- Pgp-1-) became CD4+CD8+ in less than 1 day of culture without added stimuli or growth factors. This suggested they had already received signals initiating CD4 and CD8 acquisition. However, stimulation of these precursor cells with phorbyl ester and ionomycin prevented this acquisition of CD4 and CD8. No distinct CD4-CD8+ intermediate was detected as the CD4-CD8- cells became CD4+CD8+ in the non-stimulated cultures, thus questioning the assumption that these three groups of cells are sequential steps in one lineage. In contrast to this pre-programmed acquisition of CD4 and CD8, the less mature CD4-CD8- IL-2R+ subpopulation did not progress to the CD4+CD8+ stage in culture, although it is able to develop further on intrathymic transfer. It is likely that this subpopulation represents a control point requiring specific differentiation signals for further development.

Animals↗

PubMed1986

Rapid recovery of non-hemolyzed serum and untraumatized cells by using a new method of blood defibrination in vitro.

Platelet-free cellular elements and non-hemolyzed, chemically unaltered serum are important research components of the cellular immunology laboratory. Both can be recovered from the same peripheral blood sample if it is properly defibrinated. The numbers of cells recovered from heparinized aliquots of blood from healthy donors were not significantly different from the numbers of mononuclear leukocytes, polymorphonuclear leukocytes, and erythrocytes recovered from blood samples which had been mechanically defibrinated in vitro with a stationary, cone-shaped 'TP'-like device which we here describe. Compared with serums obtained from clotted blood, or from blood defibrinated by using glass beads, we found that serums from blood defibrinated with the 'TP'-like device had the lowest detectable levels of hemoglobin, free DNA, or LDH. Serums from TP-defibrinated blood were not different from clotted serum samples with regard to the function of the classical complement pathway, the alternative complement pathway, C4 hemolytic activity, and most serum chemistries. Use of the TP-defibrinator in immunology laboratories is an ideal way to prepare blood for rapid isolation of cellular elements and non-hemolyzed serum from the same sample.

Aspartate Aminotransferases↗