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S R Carding

Publications and source records attributed to S R Carding.

At least 55 records · Page 3Linked to original sources
PubMed1993

Activation of cytokine genes in T cells during primary and secondary murine influenza pneumonia.

The patterns of cytokine mRNA expression in mice with primary or secondary influenza pneumonia have been assessed by in situ hybridization analysis of cells from both the mediastinal lymph node (MLN) and the virus-infected lung. Evidence of substantial transcriptional activity was found in all lymphocyte subsets recovered from both anatomical sites. The kinetics of cytokine mRNA expression after primary infection with an H3N2 virus were in accord with the idea that the initial response occurs in regional lymphoid tissue, with the effector T cells later moving to the lung. This temporal separation was much less apparent for the more rapid secondary response resulting from challenge of H3N2-primed mice with an H1N1 virus. Among the T cell receptor alpha/beta+ subsets, transcripts for interferon (IFN) gamma and tumor necrosis factor beta were most commonly found in the CD8+ population whereas mRNA for interleukin (IL) 4 and IL-10 was much more prevalent in CD4+ T cells. The gamma/delta T cells expressed mRNA for all cytokines tested, with IL-2, IL-4, and IFN-gamma predominating among those recovered from the inflammatory exudate. At particular time points, especially early in the MLN and late in the infected lung, the frequency of mRNA+ lymphocytes was much higher than would be expected from current understanding of the prevalence of virus-specific precursors and effectors. If this response is typical, induction of cytokine gene expression for T cells that are not responding directly to the invading pathogen may be a prominent feature of acute virus infections.

Animals↗
PubMed1993

Interferon gamma inhibits apoptotic cell death in B cell chronic lymphocytic leukemia.

The malignant, CD5+ B lymphocytes of B cell chronic lymphocytic leukemia (B-CLL) die by apoptosis in vitro. This is in contrast to the prolonged life span of the leukemic cells in vivo and likely reflects the lack of essential growth factors in the tissue culture medium. We found that interferon gamma (IFN-gamma) inhibits programmed cell death and promotes survival of B-CLL cells in culture. This effect may also be important in vivo: increased serum levels of IFN-gamma, ranging from 60 to > 2,200 pg/ml, were found in 7 of 10 B-CLL samples tested, whereas the sera of 10 healthy individuals did not contain detectable levels of this cytokine (< 20 pg/ml). High levels of IFN-gamma message were detected in RNA from T cell-depleted B-CLL peripheral blood samples by Northern blot analysis. Synthesis of IFN-gamma by B-CLL lymphocytes was confirmed by in situ hybridization and flow cytometry. The majority of B-CLL cells (74-82%) expressed detectable levels of IFN-gamma mRNA, and CD19+ B-CLL cells were labeled with anti-IFN-gamma monoclonal antibodies. These results show that IFN-gamma inhibits programmed cell death in B-CLL cells and suggest that the malignant cells are able to synthesize this cytokine. By delaying apoptosis, IFN-gamma may extend the life span of the malignant cells and thereby contribute to their clonal accumulation.

Antigens, CD↗
PubMed1993

hsp65 mRNA+ macrophages and gamma delta T cells in influenza virus-infected mice depleted of the CD4+ and CD8+ lymphocyte subsets.

The effects of depleting CD4+ and CD8+ T cells on macrophage recruitment have been analyzed for bronchoalveolar lavage (BAL) populations from mice with primary or secondary influenza pneumonia. Macrophages were characterized by both the capacity to engulf latex particles and the expression of mRNA for a 65 kD heat shock protein (hsp65). The localization of hsp65 mRNA+ cells to the pneumonic lung was greatly enhanced in the secondary response. Eliminating the CD4+ and CD8+ T cells decreased the prevalence of hsp65 mRNA+latex+ macrophages as much as seven-fold, though the frequency of latex+ cells was higher in the residual inflammatory process. The CD4-8- gamma delta T cells were also relatively enriched in the BAL from the depleted mice. However, the localization of gamma delta T cells to the pneumonic lung does not compensate either quantitatively or qualitatively for the lack of the CD4+ and CD8+ alpha beta T-cell subsets, which are responsible for activating a substantial proportion of the phagocytic cells to express transcripts of an endogenous hsp65 gene.

Animals↗
PubMed1992

Characterization of gamma delta T lymphocytes at the maternal-fetal interface.

A specific subset of gamma delta T lymphocytes bearing a V gamma 6V delta 1-encoded TCR is known to populate the normal nonpregnant murine uterine and vaginal epithelium. However, gamma delta T lymphocytes residing at the maternal-fetal interface during pregnancy have not yet been investigated. Using mAb and cytofluorographic analysis, we analyzed gamma delta TCR-bearing lymphocytes obtained from placental/decidual tissues of allogeneic (C57B1/10 X BALB/c and BALB/c X C57B1/10) pregnancies. Gestations were analyzed at several time points during the second half of pregnancy, with lymphocytes from both maternal spleen and nonpregnant C57B1/10 uteri analyzed as controls. We found that all gamma delta T lymphocytes at the maternal-fetal interface are maternally derived. Relative to the total T lymphocyte population, the percentage of gamma delta TCR-bearing T lymphocytes at the maternal-fetal interface is enriched three- to four-fold compared with maternal spleen, and twofold compared with nonpregnant uteri. Although one-third of gamma delta T lymphocytes from pregnant animals express a cell-surface marker associated with activation (IL-2R), gamma delta cells from uteri of nonpregnant mice fail to express IL-2R. In terms of absolute numbers, we estimate that reproductive-tract gamma delta T cells are increased nearly 100-fold in pregnant animals compared with nonpregnant animals. To characterize the TCR-gamma delta repertoire in the placenta/decidua, we generated 21 TCR-gamma delta-bearing hybridomas from lymphocytes in this tissue. Analysis of these hybridomas revealed at least six distinct gamma delta receptor types expressed at the maternal-fetal interface, with V gamma 6V delta 1 encoded TCR representing the predominant population. As specific resident constituents of the reproductive tract, gamma delta T lymphocytes may be involved in regulating a variety of physiologic and pathophysiologic events in reproductive biology.

Animals↗
PubMed1992

A polymerase chain reaction assay for the detection and quantitation of cytokine gene expression in small numbers of cells.

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay is described that allows the rapid detection and quantitation of mRNA encoding the cytokines interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma). Analysis of cytokine production by defined CD4+ T cell clones and the thymoma cell line EL4, demonstrates that the oligonucleotide primers used in this assay are specific for the genes encoding the individual cytokines, generating PCR products of different sizes. This allows the simultaneous and unambiguous detection of all three cytokine mRNAs in the same cDNA sample. The assay is sensitive enough to reproducibly detect cytokine mRNA expressed in as few as ten cells and requires 10,000-fold less cells for the detection of IL-2 production than that required for its detection using a conventional bioassay. Reverse transcribed mRNA is quantitated in the PCR assay by amplifying in the presence of a known amount of competitive genomic DNA (gDNA) template containing a small intron using the same primers. The PCR products obtained form the target cDNA and gDNA templates, which are distinguished by size, are processed by Southern analysis and quantitated by scanning densitometry of autoradiographs. As little as two-fold differences in cytokine mRNA can be reliably detected using this assay. We have demonstrated the successful application of this assay to the quantitation of pg amounts of IL-2 mRNA that is constitutively produced at low levels by fetal thymocytes in vivo during T cell ontogeny. The sensitivity, specificity, reliability and speed of this assay will facilitate the analysis of cytokine production in in vivo-derived or, in vitro propagated cells which are not available in sufficient numbers for analysis using more conventional molecular and biochemical assays.

Animals↗
PubMed1992

Heat shock protein Hsp60-reactive gamma delta cells: a large, diversified T-lymphocyte subset with highly focused specificity.

Previously, we detected a subset of gamma delta T cells in the newborn mouse thymus that responded to the mycobacterial heat shock protein Hsp60, as well as with what seemed to be a self-antigen. All of these cells expressed V gamma 1, most often in association with V delta 6+. It was not clear, however, whether similar, mature gamma delta cells with Hsp60 reactivity are common outside of the thymus, or rather, whether they are largely eliminated during development. From the data presented here, we estimate that gamma delta cells responding to Hsp60 comprise 10-20% of normal splenic and lymph node gamma delta T cells. Such cells, derived from adult spleen, always express a V gamma 1-J gamma 4-C gamma 4 gamma chain, although not all cells with this gamma chain show Hsp60 reactivity. Many of these V gamma 1+ cells also express V delta 6-J delta 1-C delta, though fewer than in V gamma 1+ cells from the newborn thymus. Extensive diversity is evident in both the gamma and delta chain junctional amino acids of the receptors of these cells, indicating that they may largely develop in the thymus of older animals or undergo peripheral expansion. Finally, we found that all such cells responding to both a putative self-antigen and to mycobacterial Hsp60 respond to a 17-amino acid synthetic peptide representing amino acids 180-196 of the Mycobacterium leprae Hsp60 sequence. This report demonstrates that a large subset of Hsp60-reactive peripheral lymphoid gamma delta T cells preexists in normal adult mice, all members of which respond to a single segment of this common heat shock protein.

Animals↗
PubMed1992

Analyzing the distribution of cells expressing mRNA for T cell receptor gamma and delta chains in a virus-induced inflammatory process.

Acute inflammatory processes are extremely complex, containing sets of activated cells that may be difficult to categorize. The interface between two methodologies for characterizing the involvement of gamma delta T cells, in situ hybridization to detect T cell receptor (TCR) mRNA and flow cytometric analysis of surface TCR expression, is utilized here to study the pneumonia caused by intranasal (i.n.) infection of mice with influenza A viruses. Substantial numbers of cells expressing mRNA for the gamma and delta TCR chains are present in bronchoalveolar lavage (BAL) populations obtained either late in the course of primary infection with an H3N2 virus or within a few days of secondary challenge with an H1N1 virus. The majority of the gamma delta TCR mRNA+ cells detected in FACS-separated BAL populations partition to the Thy1+ gamma delta TCR+ subset, while relatively few (less than 10%) C delta mRNA transcripts are found in cells that phagocytose latex particles. However, an additional set of gamma delta TCR mRNA+ cells is also located in a high side scatter (H-SSC) population, which stains nonspecifically with monoclonal antibodies (mAbs) and is normally gated out in the process of flow cytometric analysis. This H-SSC population tends to be enriched for cells expressing C gamma 1/2 rather than C gamma 4 mRNA. While some gamma delta TCR+ lymphocytes can be demonstrated by in vitro stimulation of the CD3 epsilon+ subset within this H-SSC population, the majority of the gamma delta T cell precursors that can be expanded in culture demonstrate a low side scatter (L-SSC) profile more characteristic of normal T lymphocytes. The possibility that subsets of activated, granular (H-SSC) alpha beta TCR+ and C gamma 1/2 mRNA+ gamma delta T cells are being missed when conventional FACS analysis is used to study this viral pneumonia is discussed.

Animals↗
PubMed1992

Extent of gamma delta T cell involvement in the pneumonia caused by Sendai virus.

The prevalence of gamma delta T cells in bronchoalveolar lavage (BAL) populations recovered from the respiratory tract of young, adult C57BL/6J mice infected intranasally (i.n.) with Sendai virus has been assessed by FACS-phenotyping, and by probing cytocentrifuge preparations for expression of TCR gamma mRNA. The surface gamma delta TCR+ set comprised from 5 to 20% of the inflammatory lymphocytes in sequential samples taken throughout the course of this nonfatal viral pneumonia. The BAL population also contained numerous cells expressing mRNA for C gamma 1/2 and C gamma 4; the C-regions were utilized for productive TCR gene rearrangement. Sorting the lymphocytes from the BAL established that greater than 90% of both the TCR gamma and TCR beta mRNA partitioned to cells with the appropriate surface TCR phenotype, while less than 7% of the TCR mRNA+ cells in the total inflammatory exudate were phagocytes that engulfed latex particles. Both the frequency and the total numbers of the gamma delta TCR+ and TCR gamma mRNA+ cells were increased in mice depleted of alpha beta T cells by in vivo treatment with mAbs to CD4 and CD8, indicating that the CD4+ and CD8+ alpha beta and CD4-8- gamma delta T cell subsets may operate independently in this virus disease. The C gamma 1/2 mRNA phenotype predominated throughout the course of the active infection, with a transition to maximal prevalence of the C gamma 4 mRNA+ set occurring very late (Day 20) in the resolving inflammatory process. Large numbers of macrophages expressing mRNA (greater than 50%) for a mammalian 65-kDa heat shock protein (hsp65), a possible target for some of the gamma delta T cells, were present early (Days 5-7) and remained at lower levels (less than 20%) thereafter. These hsp65 mRNA+ macrophages were much less apparent in BAL populations from mice depleted concurrently of the CD4+ and CD8+ T cell subsets, indicating that exposure to Sendai virus alone is not the major factor inducing the transcription of this endogenous gene. These experiments thus establish that gamma delta T cells are a minority of the infiltrating lymphocytes in Sendai virus pneumonia and provide new insights into the spectrum of hsp65 mRNA and TCR gamma mRNA expression during an inflammatory process.

Animals↗
PubMed1992

Roles of alpha beta and gamma delta T cell subsets in viral immunity.

The current status of T-cell subset involvement in viral immunity is summarized for experimental studies in mice. The immunobiology of the normal host response is discussed, with particular reference to lymphocytic choriomeningitis (LCM) and influenza. The general impression is that CD8+ cytotoxic T lymphocytes, CD4+ TH1 cells, gamma interferon, and IL-2 are of major importance, with these different components of the immune system interacting to promote an optimal response. However, experiments with a variety of virus systems indicate that there is considerable plasticity, at least in young, adult mice. Other mechanisms often compensate if a key lymphocyte subset is absent throughout the development of the immune response. Influenza-infected mice depleted of either CD4+ or CD8+ T cells clear virus and recover, though the latter may not be true for the elimination of LCM virus. Emerging information on the involvement of gamma delta T cells in viral pneumonia is summarized, but there is as yet no understanding of the biological significance (if any) of these lymphocytes in viral immunity. The point is made that alpha beta T-cell memory to viruses is long-lived, and the need for antigen persistence to maintain such memory is questioned.

Animals↗
PubMed1991

Activation status of the CD4-8- gamma delta-T cells recovered from mice with influenza pneumonia.

The role that gamma delta-T lymphocytes play in virus infections is yet to be defined. The TCR-gamma delta + cell population found late in the course of influenza pneumonia has been analyzed for ligand-dependent lytic function. These gamma delta-T cells are not constitutively cytotoxic when recovered directly from the site of virus-induced damage in the respiratory tract, although the TCR-alpha beta + population that is present concurrently contains such lytic effectors. Both sets of lymphocytes mediate cytotoxic activity after further in vitro stimulation in the presence of mAb to CD3 and low concentrations of rIL-2. Secondary stimulation in vivo with a cross-reactive influenza A virus does not lead to the emergence of a cytotoxic gamma delta-T cell population, although substantial numbers of these gamma delta-T cells express mRNA for a variety of lymphokines and cytokines. Analysis of DNA content indicates that many of the gamma delta-T cells isolated directly from the pneumonic lung are cycling. This could reflect continuing stimulation by a specific ligand, perhaps a self-component expressed at abnormally high levels in the site of virus-induced pathology. However, we could find no evidence to indicate that the gamma delta-T cells are acting to eliminate redundant components of the host response. The percentage of inflammatory macrophages and nonphagocytic cells expressing mRNA for a 65-kDa heat-shock protein (the proposed target for at least a subset of these gamma delta-T cells) is not reduced during the time that lymphocytes with mRNA for the TCR-gamma delta are present in greatest numbers. Possible alternative functions for the gamma delta-T cells are discussed.

Animals↗
PubMed1991

Regulated expression and structure of T cell receptor gamma/delta transcripts in human thymic ontogeny.

Gamma delta (gamma delta) T cells have been found in all vertebrates examined, yet their function in vivo remains unknown. Because gamma delta T cell receptors are related to immunoglobulin, and because they are encoded by rearranging, multi-gene families, the receptors are thought to be antigen recognition molecules. However, a capacity to recognize naturally diverse antigens has not yet been shown. In this work, the expression and structure of human gamma delta transcripts have been examined in the fetal and early post-natal thymus. The data indicate that many gamma and delta genes are rearranged and expressed throughout ontogeny, but that as ontogeny proceeds, quite dramatic changes occur in the patterns of gene expression and rearrangement. In particular, receptors encoded by early to mid-gestation fetal thymic transcripts would be of quite restricted diversity. Only later in ontogeny can receptors of substantial diversity be generated. These properties are very similar to the patterns of gamma delta gene activation in the mouse, and they serve to reiterate similarities both in gene rearrangement and in gamma delta across vertebrate species.

Base Sequence↗
PubMed1991

Cytokines in T-cell development.

The thymus provides a unique environment for the development of T cells, supporting both precursor cell proliferation and differentiation. The control of these processes is unknown but they may be mediated by cytokines, or other soluble factors, or by interactions with specific elements of the thymic stroma. Here, Simon Carding, Adrian Hayday and Kim Bottomly describe cellular, immunochemical and molecular studies of the production and action of cytokines within the human and mouse thymus and demonstrate their essential role in T-cell development.

Animals↗
PubMed1990

Late dominance of the inflammatory process in murine influenza by gamma/delta + T cells.

The inflammatory response in the lungs of mice infected with an influenza A virus consists largely of macrophages and CD3+ T cells. Most T lymphocytes recovered before day 7 after infection express mRNA for the T cell receptor alpha/beta (TCR-alpha/beta), while TCR-gamma/delta mRNA+ cells are found at much higher frequency over the next 7 d. The predominant surface phenotype for the TCR-gamma/delta mRNA+ population is CD3+4-8-TCR-alpha/beta-. Some lymphocytes expressing all the known V gamma genes are found in the inflammatory exudate, but V gamma 2+/V gamma 1+ and V gamma 4+ T cells are present at highest frequency. The response is staged, with maximal numbers of V gamma 4+ cells occurring on day 10 after infection, while the predominant phenotype on day 13 is V gamma 2/V gamma 1+. The emerging peak in numbers of V gamma 4+ lymphocytes is paralleled by increasing numbers of macrophages expressing hsp mRNA. The later maxima found for the V gamma 2+/V gamma 1+ T cells is consistent with the possibility that at least some of these lymphocytes are responding to the hsp+ cells and are functioning to resolve the inflammatory process.

Animals↗
PubMed1990

Characterization of gamma/delta T cell clones isolated from human fetal liver and thymus.

The origin and development of T cells bearing gamma/delta T cells receptors (TcR) has been extensively studied in the mouse. By contrast, little is known about development patterns and diversity of the human gamma/delta T cell lineage. To study the repertoire of human gamma/delta+ T cells during T cell ontogeny, we have isolated clonal populations of gamma/delta+ T cells from 14-week fetal thymus and liver and characterized the molecular composition of their TcR. The technique of in situ hybridization was used to identify cells expressing TcR genes in fetal liver and thymus. A panel of clones representative of developing T cell populations found in vivo was subsequently isolated from both tissues and clones expressing cell surface gamma/delta receptors were identified. Although both the liver-derived gamma/delta+ T cell clone, L2, and the thymus-derived gamma/delta+ T cell clone, T6, had similar cell surface phenotypes, namely CD3+, CD7+, CD45+ and CD8-, their reactivity with anti-CD2 and -CD4 antibodies was different. L2 was CD2high, CD4- whereas T6 was CD2low, CD4low. Both clones possessed effector functions similar to those of adult T cells as demonstrated by the synthesis and secretion of cytokines in response to stimulation through the CD3/TcR complex. Analysis of the TcR composition of the fetal clones showed both clones to possess similar or identical gamma chain components, C gamma 1, J gamma 1/2, V gamma 8, and both utilize V delta gene segments other than V delta 1. This TcR genotype has not been previously reported in the analysis of adult gamma/delta+ T cells. Our studies have identified a unique population of human gamma/delta+ T cells that may be derived extrathymically and appear to be preferentially and perhaps transiently expressed during fetal life.

Biological Factors↗
PubMed1990

Developmentally regulated fetal thymic and extrathymic T-cell receptor gamma delta gene expression.

The gamma delta T-cell receptor (TCR) is the first TCR to be expressed in ontogeny in all vertebrates in which it has been examined thoroughly. Murine gamma delta cell-surface protein is detected by the fourteenth day of gestation. In this work, the activation of gamma delta RNA has been studied. Data indicate that the first TCR protein to appear in the thymus is encoded by gamma genes that are activated after cells colonize the thymus. However, the sequential appearance of different gamma delta TCR proteins during thymic ontogeny cannot be readily explained by differential temporal activation of V gamma genes in the thymus. There are distinct patterns of gamma and delta gene expression during fetal liver development and in the fetal gut (or tissue associated with it). Cells apparent in the liver of mice at birth express gamma delta cell-surface protein, but they disappear from the liver very soon afterward. One V gamma gene is rearranged and expressed prethymically. In addition, gamma gene expression is detectable in the livers of newborn athymic mice. Together, these observations indicate a thymic-independent pathway of activation of TCR genes.

Animals↗