Persistent HIV-1-specific CTL clonal expansion despite high viral burden post in utero HIV-1 infection.
(17/2292)
To address the issue of clonal exhaustion in humans, we monitored HLA class I-restricted, epitope-specific CTL responses in an in utero HIV-1-infected infant from 3 mo through 5 years of age. Serial functional CTL precursor assays demonstrated persistent, vigorous, and broadly directed HIV-1 specific CTL activity with a dominant response against an epitope in HIV-1 Gag-p17 (SLYNTVATL, aa 77-85). A clonal CTL response directed against the immunodominant, HLA-A*0201-restricted epitope was found to persist over the entire observation period, as shown by TCR analysis of cDNA libraries generated from PBMC. The analysis of autologous viral sequences did not reveal any escape mutations within the targeted epitope, and viral load measurement indicated ongoing viral replication. Furthermore, inhibition of viral replication assays indicated that the epitope was properly processed from autologous viral protein. These data demonstrate that persistent exposure to high levels of viral Ag does not necessarily lead to clonal exhaustion and that epitope-specific clonal CTL responses induced within the first weeks of life can persist for years without inducing detectable viral escape variants. (+info)
Cell-surface expression and alloantigenic function of a human nonclassical class I molecule (HLA-E) in transgenic mice.
(18/2292)
We have introduced the gene (E*01033) encoding the heavy chain of the human nonclassical MHC class I Ag, HLA-E, into the mouse genome. Two founder mice carry a 21-kb fragment, the others bear an 8-kb fragment. Each of the founder mice was mated to mice of an already established C57BL/10 transgenic line expressing human beta2-microglobulin (beta2m). Cell surface HLA-E was detected on lymph node cells by flow cytometry only in the presence of endogenous human beta2m. However, HLA-E-reactive mouse CTL (H-2-unrestricted) lysed efficiently the target cells originating from HLA-E transgenic mice without human beta2m, showing that the HLA-E protein can be transported to the cell surface in the absence of human beta2m, presumably by association with murine beta2m. Rejection of skin grafts from HLA-E transgenic mice demonstrates that HLA-E behaves as a transplantation Ag in mice. HLA-E transgenic spleen cells are effective in stimulating an allogeneic CTL response in normal and human classical class I (HLA-B27) transgenic mice. Furthermore, results from split-well analysis indicate that the majority of the primary in vivo-induced CTL recognizes HLA-E as an intact molecule (H-2-unrestricted recognition) and not as an HLA-E-derived peptide presented by a mouse MHC molecule, although a small fraction (ranging from 4 to 21%) of the primary in vivo-induced CTL is able to recognize HLA-E in an H-2-restricted manner. Based on these observations, we conclude that HLA-E exhibits alloantigenic properties that are indistinguishable from classical HLA class I molecules when expressed in transgenic mice. (+info)
Induction of primary human CD8+ T lymphocyte responses in vitro using dendritic cells.
(19/2292)
The ability of two different human professional APCs, specifically macrophages (Mphi) and dendritic cells (DC), to stimulate primary responses in human CD8+ T lymphocytes was examined using both allogeneic and Ag-pulsed autologous APCs. CTL responses in CD8+ T lymphocytes isolated from HIV-uninfected donors were evaluated against six different HIV epitopes that are restricted by four different HLA alleles using autologous human PBMC-derived Mphi and DCs for primary stimulation. In a side-by-side experiment, immature DCs, but not Mphi, were able to prime a CTL response against the B14-restricted p24gag 298-306 epitope; mature DCs were also able to prime a response against this epitope. In addition, DCs were capable of priming CD8+ CTL responses against the B8-restricted p24gag 259-267 epitope. In contrast, Mphi were unable to prime strong CTL responses against other epitopes. Since the Ag-specific cytotoxic responses required subsequent rounds of restimulation before they could be detected, the ability of the allogeneic Mphi and DCs to directly prime CD8+ T lymphocyte responses without subsequent restimulation was examined. Similar to the aforementioned peptide-specific results, DCs were more efficient than Mphi in priming both allogeneic proliferative and cytotoxic responses in human CD8+ T lymphocytes. Collectively, these results promote an enhanced status for DCs in the primary stimulation of human CD8+ T lymphocytes. (+info)
Indomethacin inhibits circulating PGE2 and reverses postexercise suppression of natural killer cell activity.
(20/2292)
Natural killer (NK) cells are important in combating viral infections and cancer. NK cytolytic activity (NKCA) is often depressed during recovery from strenuous exercise. Lymphocyte subset redistribution and/or inhibition of NK cells via soluble mediators, such as prostaglandin (PG) E2 and cortisol, are suggested as mechanisms. Ten untrained (peak O2 consumption = 44.0 +/- 3.5 ml. kg-1. min-1) men completed at 2-wk intervals a resting control session and three randomized double-blind exercise trials after the oral administration of a placebo, the PG inhibitor indomethacin (75 mg/day for 5 days), or naltrexone (reported elsewhere). Circulating CD3(-)CD16(+)/56(+) NK cell counts, PGE2, cortisol, and NKCA were measured before, at 0.5-h intervals during, and at 2 and 24 h after a 2-h bout of cycle ergometer exercise (65% peak O2 consumption). During placebo and indomethacin conditions, exercise induced significant (P < 0.0001) elevations of NKCA (>100%) and circulating NK cell counts (>350%) compared with corresponding control values. With placebo treatment, total NKCA was suppressed (28%; P < 0.05) 2 h after exercise, and a postexercise elevation (36%; P = 0.02) of circulating PGE2 was negatively correlated (r = 0.475, P = 0.03) with K-562 tumor cell lysis. NK counts were unchanged in the postexercise period, but at this stage CD14(+) monocyte numbers were elevated (P < 0.0001). Indomethacin treatment eliminated the postexercise increase in PGE2 concentration and completely reversed the suppression of total and per CD16(+)56(+) NKCA 2 h after exercise. These data support the hypothesis that the postexercise reduction in NKCA reflects changes in circulating PGE2 rather than a differential lymphocyte redistribution. (+info)
A phage-displayed mimotope inhibits tumour necrosis factor-alpha-induced cytotoxicity more effectively than the free mimotope.
(21/2292)
A phage-displayed peptide library was screened by direct interaction with human tumour necrosis factor-alpha (TNF-alpha) to identify novel antagonistic molecules of its biological activities. After several rounds of affinity selection, a phage displaying a mimotope sequence was shown to strongly inhibit, in a dose-dependent fashion, both mouse and human TNF-alpha-mediated cytotoxicity in L929 cells. The identified mimotope did not bear any sequence homology to the primary structures of the extracellular domains of either the 55 000 MW or the 75 000 MW TNF-alpha receptors, suggesting that it represents or mimics a conformational epitope involved with binding to TNF-alpha. The free 15-mer mimotope weakly inhibited TNF-alpha-induced cytotoxicity in vitro, and it did not bind to TNF-alpha as assessed by surface plasmon resonance, demonstrating the importance of mimotope presentation for its biological activities. In conclusion, this study highlights the potential of random combinatorial peptide libraries for the identification of novel inhibitors, which may serve as important tools in research that could lead to the development of TNF-alpha antagonists with therapeutic potential. (+info)
Enhancing the immunotherapeutic potential of mycobacteria by transfection with tumour necrosis factor-alpha.
(22/2292)
In an attempt to enhance the anti-tumour properties of mycobacteria we have developed recombinant forms of Mycobacterium smegmatis which express and secrete biologically active human tumour necrosis factor-alpha (TNF-alpha). This was achieved by transfecting M. smegmatis using shuttle plasmids incorporating the cDNA sequence for the human TNF-alpha mature peptide. In vitro experiments on a panel of human bladder tumour cell lines (EJ18, MGH-U1, RT4, RT112) indicate that our genetically modified mycobacteria are more effective than wild-type at inducing or up-regulating the expression of intracellular adhesion molecule-1 and the secretion of an array of proinflammatory cytokines [interleukin-1 (IL-1), IL-6, IL-8, granulocyte-macrophage colony-stimulating factor]. We have also demonstrated increased adhesion molecule and cytokine expression in response to mycobacteria transfected with vector containing no gene insert. However, this was not as pronounced as that observed following tumour cell stimulation by the TNF-alpha-transfected strain. In contrast, in three out of four tumour cell lines all M. smegmatis strains were found to down-regulate the secretion of the anti-inflammatory cytokine transforming growth factor-beta1. Our studies have also confirmed that M. smegmatis is a powerful inhibitor of bladder tumour cell growth and revealed that its antiproliferative potency is enhanced by transfecting with human TNF-alpha and, to a lesser extent, with vector alone. All M. smegmatis strains were effective in the activation of peripheral blood leucocyte cultures. However, no differences were observed in the ability of the TNF-alpha-transfected, mock-transfected and wild-type mycobacteria to induce tumour cell killing activity. These results suggest that the immunomodulatory effects of M. smegmatis can be enhanced by transfection with vectors which allow the secretion of human TNF-alpha, thus increasing mycobacterial immunotherapeutic potential. (+info)
Specific lysis of melanoma cells by receptor grafted T cells is enhanced by anti-idiotypic monoclonal antibodies directed to the scFv domain of the receptor.
(23/2292)
Malignant transformation of melanocytes is frequently associated with abnormalities in antigen processing and in human leukocyte antigen class I antigen expression. Here, we evaluated a human leukocyte antigen class I antigen-independent approach to target cytotoxic T lymphocytes to melanoma cells by grafting cytotoxic T lymphocytes with a chimeric receptor that consists of both a domain binding to high molecular weight-melanoma associated antigen and a cellular activation domain. The binding domain is a single-chain antibody fragment (scFv) derived from the monoclonal anti-high molecular weight-melanoma associated antigen antibody 763.74 by phage display techniques. The cellular activation domain is the signaling unit of the FcepsilonRI receptor gamma chain. Both domains constitute the chimeric receptor scFv763.74-gammaR. Cytotoxic MD45 T cells grafted with the scFv763.74-gammaR receptor bind specifically to high molecular weight-melanoma associated antigen-positive melanoma cells and lyse melanoma cells in a human leukocyte antigen class I independent fashion. Pre-incubation of receptor grafted T cells with immobilized anti-idiotypic (id) monoclonal antibody MK2-23 binding to the scFv domain of the receptor enhanced the lysis of melanoma cells indicating that the specific cytolytic activity of receptor grafted T cells can be increased by costimulation with cross-linked anti-idiotypic monoclonal antibodies that recognize the antigen binding domain of the chimeric receptor. (+info)
Quantitative and qualitative effects of cyclophosphamide administration on circulating polymorphonuclear leucocytes.
(24/2292)
The effect of cyclophosphamide (CY) on the absolute numbers and function of polymorphonuclear leucocytes (PMN) surviving in the circulation following either a single dose (100 mg/kg, i.p.) or daily administration (20 mg/kg, i.p., for 5 days) was studied in the guinea-pig. The quantitative effect of CY on peripheral blood leucocytes was assessed by measuring the absolute numbers of neutrophils, lymphocytes, and monocytes daily for 5 days following the initial injection of CY. The qualitative effects of CY on PMN function were determined by measuring the ability of these cells to function as killer cells. The two functional assays employed were the PMN-mediated PHA-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) assays against chicken erythrocyte targets. Both regimens of CY administration produced an equivalent degree of leukopenia 5 days after the initial injection with disproportionately severe neutropenia (less than 300 PMN/mm3). However, neither regimen of CY administration produced a significant decrease in cytotoxic effector function as measured through a wide range of effector to target cell ratios, PHA concentrations, and antiserum dilutions. These findings have clinical relevance in that they demonstrate the dichotomy between the quantitative and qualitative effects of (CY) on PMNs in that CY administration can dramatically decrease the absolute numbers of circulating polymorphonuclear leucocytes while leaving intact certain effector cell functional capabilities of those PMN surviving in the circulation during drug administration. (+info)