Differential expression of Fas ligand in Th1 and Th2 cells is regulated by early growth response gene and NF-AT family members.

Inducible expression of Fas ligand (CD95 ligand) by activated T cells and the resulting apoptosis of CD95-bearing cells is a critical component of peripheral T cell homeostasis and cytotoxic effector mechanisms. Transcriptional control of the expression of Fas ligand has been attributed to a number of factors, including early growth response gene 2 (Egr2), Egr3, Sp1, and NF-AT, although a direct contribution of NF-AT is controversial. The present study confirms a role for Egr factors and indicates that NF-AT is essential for optimal expression of murine Fas ligand through a direct interaction with an NF-AT consensus element. The role of these factors was further defined by studying the differential expression of Fas ligand in Th1 and Th2 lines derived from DO11.10 TCR transgenic mice. EMSA analyses of a composite Egr/NF-AT site showed recruitment of Sp1 to this site in Th2 cells, but not in Th1 cells. Furthermore, gel-shift analyses demonstrated the binding of Egr1, 2, and 3 in Th2 cells and Egr1 and 2, but not Egr3 in Th1 cells at a known Egr site. Northern analysis corroborated the lack of Egr3 in Th1 cells. Differential usage of these transcription factors by Th1 and Th2 cells suggests a potential mechanism underlying the differential expression of Fas ligand by distinct T cell lineages.

Stability in the HIV vDNA pool in peripheral CD4+ T cells of untreated patients by single tube quantitative PCR.

HIV infection leads to loss of CD4 T cells and development of AIDS in most individuals without treatment. While disease progression during HIV infection correlates with the plasma viral load, much less is known about the levels of HIV vDNA. This paper describes the development and validation of a sensitive, quantitative PCR assay for the assessment of HIV vDNA. The system uses novel single tube, multiply competitive PCR technology, which allows five-point competitor competition in a single PCR reaction. The reproducibility and performance characteristics of the assay are extensively studied, which indicate that the system performs well in high DNA backgrounds. Using this assay system on a cohort of protease naïve patients, HIV vDNA was assessed from PBMCs over an average follow-up period of 5 years. The data indicate that the HIV vDNA pool does not appreciably accumulate over the follow-up period, with many of the patients followed for up to 8 years. A reliable, quantitative assessment of vDNA pools will allow a better understanding of the dynamics of HIV pathogenesis.

Evaluation of distinct blood lymphocyte populations in human immunodeficiency virus type 1-infected subjects in the absence or presence of effective therapy.

Virus reservoirs can persist in human immunodeficiency virus type 1 (HIV-1)-infected subjects despite effective plasma virus suppression. To compare viral dynamics in the absence and presence of antiretroviral therapy, blood mononuclear cells from 19 subjects with high plasma RNA levels and 18 subjects following prolonged virus suppression were examined, by use of in situ hybridization, to detect virus RNA expression before and after in vitro T cell activation. This approach reveals circulating lymphocytes expressing HIV-1 RNA before activation and an increase in cells with detectable HIV-1 RNA transcription after in vitro activation. The frequencies of these 2 cell populations are strongly correlated with plasma virus load and appear to be stable once a new steady state is established during therapy. The frequency of viral RNA-positive cells is equivalent to the frequency of cells that produce infectious virus. Thus, in HIV-1-infected subjects there are distinct virus reservoirs comprising both latent and replication-active cells.

Interleukin-2 expression by a subpopulation of primary T cells is linked to enhanced memory/effector function.

Single cell studies have identified intraclonal heterogeneity of cytokine production by activated T cells. To investigate implications of cytokine heterogeneity for cell fate, an interleukin (IL)-2 promoter-green fluorescent protein (GFP) reporter transgenic model was developed to track IL-2+ and IL-2- T cells during differentiation from naive precursors. Antigen-activated IL-2+ and IL-2- cells had comparable proliferative capacities in primary responses. However, T cells that expressed IL-2 in primary responses demonstrated enhanced antigenic sensitivity and increased expression of effector cytokines in secondary responses in vitro and in vivo. Thus, heterogeneity of activation during a primary response translates into heterogeneous secondary responses, in which enhanced memory/effector function is linked to cells that previously exceeded an activation threshold associated with IL-2 gene transcription.

Characterization of an avian (Gallus gallus domesticus) TCR alpha delta gene locus.

Mammalian TCR delta genes are located in the midst of the TCR alpha gene locus. In the chicken, one large V delta gene family, two D delta gene segments, two J delta gene segments, and one C delta gene have been identified. The TCR delta genes were deleted on both alleles in alpha beta T cell lines, thereby indicating conservation of the combined TCR alpha delta locus in birds. V alpha and V delta gene segments were found to rearrange with one, both or neither of the D delta segments and either of the two J delta segments. Exonuclease activity, P-addition, and N-addition during VDJ delta rearrangement contributed to TCR delta repertoire diversification in the first embryonic wave of T cells. An unbiased V delta 1 repertoire was observed at all ages, but an acquired J delta 1 usage bias occurred in the TCR delta repertoire. The unrestricted combinatorial diversity of relatively complex TCR gamma and delta loci may contribute to the remarkable abundance of gamma delta T cells in this avian representative.

Detection of Chlamydia trachomatis endocervical infections by ligase chain reaction versus ACCESS Chlamydia antigen assay.

Ligase chain reaction (LCR) was compared with ACCESS immunoassay for detection of chlamydial infections in females. Despite efforts to improve ACCESS performance by evaluation of specimens that were in the test performance "grey zone," LCR remained more sensitive and was less expensive to perform. ACCESS had a sensitivity of 83.9%, a specificity of 99.7%, a positive predictive value of 96.3%, and a negative predictive value of 98.5%.

Initial increase in blood CD4(+) lymphocytes after HIV antiretroviral therapy reflects redistribution from lymphoid tissues.

Previous studies proposed a dynamic, steady-state relationship between HIV-mediated cell killing and T-cell proliferation, whereby highly active antiretroviral therapy (HAART) blocks viral replication and tips the balance toward CD4(+) cell repopulation. In this report, we have analyzed blood and lymph node tissues obtained concurrently from HIV-infected patients before and after initiation of HAART. Activated T cells were significantly more frequent in lymph node tissue compared with blood at both time points. Ten weeks after HAART, the absolute number of lymphocytes per excised lymph node decreased, whereas the number of lymphocytes in the blood tended to increase. The relative proportions of lymphoid subsets were not significantly changed in tissue or blood by HAART. The expression levels of mRNA for several proinflammatory cytokines (IFN-gamma, IL-1beta, IL-6, and macrophage inflammatory protein-1alpha) were lower after HAART. After therapy, the expression of VCAM-1 and ICAM-1 -- adhesion molecules known to mediate lymphocyte sequestration in lymphoid tissue -- was also dramatically reduced. These data provide evidence suggesting that initial increases in blood CD4(+) cell counts on HAART are due to redistribution and that this redistribution is mediated by resolution of the immune activation that had sequestered T cells within lymphoid tissues.

Constant mean viral copy number per infected cell in tissues regardless of high, low, or undetectable plasma HIV RNA.

Quantitative analysis of the relationship between virus expression and disease outcome has been critical for understanding HIV-1 pathogenesis. Yet the amount of viral RNA contained within an HIV-expressing cell and the relationship between the number of virus-producing cells and plasma virus load has not been established or reflected in models of viral dynamics. We report here a novel strategy for the coordinated analysis of virus expression in lymph node specimens. The results obtained for patients with a broad range of plasma viral loads before and after antiretroviral therapy reveal a constant mean viral (v)RNA copy number (3.6 log10 copies) per infected cell, regardless of plasma virus load or treatment status. In addition, there was a significant but nonlinear direct correlation between the frequency of vRNA+ lymph node cells and plasma vRNA. As predicted from this relationship, residual cells expressing this same mean copy number are detectable (frequency <2/10(6) cells) in tissues of treated patients who have plasma vRNA levels below the current detectable threshold (<50 copies/ml). These data suggest that fully replication-active cells are responsible for sustaining viremia after initiation of potent antiretroviral therapy and that plasma virus titers correlate, albeit in a nonlinear fashion, with the number of virus-expressing cells in lymphoid tissue.

T cell receptor gene deletion circles identify recent thymic emigrants in the peripheral T cell pool.

Progenitor cells undergo T cell receptor (TCR) gene rearrangements during their intrathymic differentiation to become T cells. Rearrangements of the variable (V), diversity (D), and joining (J) segments of the TCR genes result in deletion of the intervening chromosomal DNA and the formation of circular episomes as a byproduct. Detection of these extrachromosomal excision circles in T cells located in the peripheral lymphoid tissues has been viewed as evidence for the existence of extrathymic T cell generation. Because all of the T cells in chickens apparently are generated in the thymus, we have employed this avian model to determine the fate of the V(D)J deletion circles. In normal animals we identified TCR Vgamma-Jgamma and Vbeta-Dbeta deletion circles in the blood, spleen, and intestines, as well as in the thymus. Thymectomy resulted in the gradual loss of these DNA deletion circles in all of the peripheral lymphoid tissues. A quantitative PCR analysis of Vgamma1-Jgamma1 and Vbeta1-Dbeta deletion circles in splenic gamma delta and Vbeta1(+) alphabeta T cells indicated that their numbers progressively decline after thymectomy with a half-life of approximately 2 weeks. Although TCR deletion circles therefore cannot be regarded as reliable indicators of in situ V(D)J rearrangement, measuring their levels in peripheral T cell samples can provide a valuable index of newly generated T cells entering the T cell pool.

Kinetics of T cell receptor beta, gamma, and delta rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44(+)CD25(+) Pro-T thymocytes.

We performed a comprehensive analysis of T cell receptor (TCR) gamma rearrangements in T cell precursors of the mouse adult thymus. Using a sensitive quantitative PCR method, we show that TCRgamma rearrangements are present in CD44(+)CD25(+) Pro-T thymocytes much earlier than expected. TCRgamma rearrangements increase significantly from the Pro-T to the CD44(-)CD25(+) Pre-T cell transition, and follow different patterns depending on each Vgamma gene segment, suggesting that ordered waves of TCRgamma rearrangement exist in the adult mouse thymus as has been described in the fetal mouse thymus. Recombinations of TCRgamma genes occur concurrently with TCRdelta and D-Jbeta rearrangements, but before Vbeta gene assembly. Productive TCRgamma rearrangements do not increase significantly before the Pre-T cell stage and are depleted in CD4(+)CD8(+) double-positive cells from normal mice. In contrast, double-positive thymocytes from TCRdelta-/- mice display random proportions of TCRgamma rearranged alleles, supporting a role for functional TCRgamma/delta rearrangements in the gammadelta divergence process.

Heterogeneity in the clonal T cell response. Implications for models of T cell activation and cytokine phenotype development.

The T cell can be defined in the context of two properties--the recognition specificity of the T cell receptor (TCR) heterodimer and the functional response of the T cell after TCR stimulation. Once a particular TCR heterodimer is expressed and successfully selected during thymic development, the antigen specificity is fixed for all the clonal progeny of that cell. In contrast, the potential functional responses that may be generated in response to specific antigen in the postthymic environment are quite extensive. These range from programmed cell death to initiation of alternate programs of phenotype development that generate effector populations with distinct cytokine expression patterns and regulatory properties. Recent advances in analytical methods that have permitted multiparametric characterizations of the T cell response at the single cell, rather than population level, have necessitated a modified view of T cell activation and the clonal T cell response, and have generated new insights into the regulation of immunity. In this brief review, we highlight studies that have characterized heterogeneity of the CD4+ T cell clonal response based on single-cell analyses, and discuss implications for models of T cell activation and cytokine phenotype development.

Effect of lipopolysaccharide and inflammatory cytokines on interleukin-6 production by healthy human gingival fibroblasts.

Cytokines are hormone-like proteins which mediate and regulate inflammatory and immune responses. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) and inflammatory cytokines on regulation of interleukin-6 (IL-6) production by human gingival fibroblasts (HGF). The HGF cell lines used in this study, H-CL and F-CL, were established by the explant technique from healthy gingival tissue. Cultured cells were grown to confluency and incubated with various concentrations of LPS from Escherichia coli or Porphyromonas gingivalis or with the recombinant human cytokine tumor necrosis factor alpha (TNF-alpha), IL-1alpha, or IL-1beta. Culture supernatants were collected at various times and assessed for IL-6 production by enzyme-linked immunosorbent assay. Total RNA was isolated from the harvested cells and used to assess levels of IL-6 mRNA by the RNase protection assay. Both LPS preparations induced IL-6 production (1 to 4 ng of IL-6 per ml) by both HGF cell lines. Although TNF-alpha stimulated IL-6 production by HGF, > 10-fold-larger amounts were induced with IL-1alpha and IL-1beta. Furthermore, the addition of both IL-1alpha and TNF-alpha to cultured cells resulted in approximately 600- to 800-fold-higher levels of IL-6 than seen in control cultures, suggesting that these cytokines synergistically induced IL-6 production by HGF. IL-6 message in cultured cells was upregulated 20-fold by TNF-alpha, 1,000-fold by IL-1alpha and IL-1beta, and 1,400-fold by IL-1alpha plus TNF-alpha. IL-1alpha and TNF-alpha alone upregulate IL-6 production in a dose- and time-dependent fashion. The addition of IL-1alpha and TNF-alpha to cultured HGF cells resulted in a synergistic induction of IL-6 after 8 h of incubation and when greater than 10 pg of this combination per ml was used. Our studies show that inflammatory cytokines are hundreds of times more potent than LPS in stimulating IL-6 production by HGF.

Identification of a DNA segment exhibiting rearrangement modifying effects upon transgenic delta-deleting elements.

Control of the rearrangement and expression of the T cell receptor alpha and delta chains is critical for determining T cell type. The process of delta deletion is a candidate mechanism for maintaining separation of the alpha and delta loci. Mice harboring a transgenic reporter delta deletion construct show alpha/beta T cell lineage-specific use of the transgenic elements. A 48-basepair segment of DNA, termed HPS1A, when deleted from this reporter construct, loses tight lineage-specific rearrangement control of transgenic elements, with abundant rearrangements of transgenic delta-deleting elements now in gamma/delta T cells. Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay. DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci. These data are consistent with delta deletion playing an important role in maintaining separate TCR alpha and delta loci.

A novel gene therapy strategy for elimination of prostate carcinoma cells from human bone marrow.

We report a novel means to purge bone marrow of a specific subset of prostate carcinoma cells based on transductional and genetic selectivity. Using both adenovirus-polylysine-DNA complexes and E1A/B-deleted replication-deficient adenoviruses, we have demonstrated a transductional preference of these vectors for the prostate carcinoma cell lines DU 145, LNCaP, and PC-3 over primary human bone marrow cells and the leukemia cell line KG-1. We have also shown a genetic selectivity of an anti-erbB-2 intracellular single-chain antibody (sFv) encoding adenovirus, Ad21, for the erbB-2-positive prostate carcinoma cell lines DU 145 and LNCaP. Delivery of Ad21 resulted in cytotoxicity to the DU 145 and LNCaP, but not PC-3, cell lines and reduced the clonogenic capacity of DU 145 cells cultured alone or mixed with various ratios of irradiated human bone marrow. Finally, quantitative, competitive reverse transcription polymerase chain reaction (QC-RT-PCR) analysis demonstrated that Ad21 could effectively reduce DU 145 and erbB-2-positive primary prostate tumor contamination in bone marrow cultures. Delivery of Ad21 had no effect on the ability of progenitor cells to form colonies. These results suggest that an anti-erbB-2 sFv-encoding adenoviral vector is efficacious for removal of erbB-2-positive prostate carcinoma cells from human bone marrow, and demonstrates a novel method for ex vivo genetic purge of malignant cells from bone marrow for autologous bone marrow transplantation (ABMT) therapy.

Quantitative assessment of inflammatory cytokine gene expression in chronic adult periodontitis.

Adult periodontitis is a chronic destructive disease characterized by an interaction between gram-negative bacteria and the host inflammatory response. Microbial substances such as lipopolysaccharide can activate host cells, e.g., macrophages, fibroblasts and keratinocytes, to secrete proinflammatory cytokines including tumor necrosis factor alpha and interleukin 1 beta (IL-1 beta). This study examined the hypothesis that periodontitis tissue contains increased levels of cytokines that promote osseous and connective tissue destruction. To test this hypothesis, diseased and healthy gingival biopsies were examined for differences in the expression of cytokine mRNA for the pro-inflammatory cytokines tumor necrosis factor alpha and IL-1 beta and the anti-inflammatory cytokine IL-1ra using quantitative reverse transcriptase polymerase chain reaction and in situ hybridization methods. The levels of tumor necrosis factor alpha and IL-1ra mRNA were shown to be significantly higher in diseased than healthy tissues. Additionally, a significantly correlated expression of IL-1 beta and IL-1ra mRNA was seen in all tissue examined. Analysis of tissue sections by immunohistochemical and in situ hybridization techniques revealed a mononuclear cell infiltrate that consisted of a higher average number of cells staining positive for tumor necrosis factor alpha mRNA, CD14, and CD3 in the diseased than healthy tissues. Although both diseased and healthy tissues expressed IL-1 beta and IL-1ra mRNA in the epithelium, the diseased tissue biopsies expressed more IL-1 beta and IL-1ra mRNA in the connective tissue. These results implicate the potential involvement of both the pro- and anti-inflammatory cytokines in the regulation of the chronic inflammatory disease adult periodontitis.

The effects of dihydrotestosterone on the expression of p185(erbB-2) and c-erbB-2 mRNA in the prostatic cell line LNCaP.

The c-erbB-2 proto-oncogene encodes a 185000 molecular weight protein (p185(erbB-2)) which shares structural homology with the epidermal growth factor (EGF) receptor. We examined the effects of dihydrotestosterone (DHT) on the expression of p185(erbB2) and c-erbB-2 mRNA in the human malignant prostatic cell line LNCaP. LNCaP cells grown in steroid-depleted media were treated with DHT (10(-11)-10(-6) M) for 48 h and p185(erbB-2) expression was determined by Western blotting and immunoprecipitation of 35S-methionine labelled p185(erbB-2). c-erbB-2 mRNA levels were determined using a competitive quantitative reverse transcription-polymerase chain reaction (RT-PCR) based technique. DHT at concentrations of 10(-9) M or greater resulted in decreased expression of p185(erbB-2). In contrast, DHT at these levels stimulated EGF receptor protein expression and cellular proliferation. c-erbB-2 mRNA levels declined to 30-50% of control levels following treatment with DHT of 10(-10) M or greater. Furthermore, the inhibitory effects on c-erbB-2 mRNA were rapid, occurring within 6-12 h of treatment. In summary, these results demonstrate that DHT, at concentrations that stimulate cell growth, inhibits the expression of p185(erbB-2) and c-erbB-2 mRNA.

Interferon-gamma differentially regulates antigen-processing functions in distinct endocytic compartments of macrophages with constitutive expression of class II major histocompatibility complex molecules.

RAW264.7 cells were transfected to express constitutively the murine class II major histocompatibility complex (MHC-II) molecule, I-Ak. The resulting RAW.Ak cells presented HEL(46-61) peptide to 3A9 T hybridoma cells, but they were unable to process and present HEL protein in their resting state. However, IFN-gamma stimulation induced the ability of RAW.Ak to process and present HEL protein, with little effect on their ability to present HEL(46-61) peptide. Antigen catabolism showed little change with IFN-gamma stimulation, suggesting that the production of peptides was not the regulated step in the processing pathway. Furthermore, HEL(46-61) peptide delivered directly into lysosomes by acid-resistant liposomes was also presented only upon IFN-gamma stimulation, while the presentation of peptides delivered into endosomes by acid-sensitive liposomes showed a lesser dependence on IFN-gamma stimulation. Thus, IFN-gamma regulated the ability of peptides delivered into certain lysosomal compartments to meet with MHC-II molecules and form peptide-MHC complexes, or to transport subsequently to the plasma membrane. Two other antigens, ribonuclease A and haemoglobin, were processed by RAW.Ak cells without IFN-gamma stimulation, suggesting that these antigens could be processed by different mechanisms, perhaps in earlier endocytic compartments. Thus, different antigens may be processed in distinct endocytic compartments, and an IFN-gamma-regulated mechanism controls the rescue of peptides from lysosomal compartments for presentation at the plasma membrane.

A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell.