Innate immune functions of human gammadelta T cells.

gammadelta T cells expressing the Vgamma9Vdelta2 T cell receptor (TCR) account for 1-10% of CD3(+) peripheral blood T lymphocytes. Vgamma9Vdelta2 T cells use their TCR as a pattern recognition receptor to sense the presence of infection through specific recognition of intermediates of the microbial non-mevalonate pathway of isoprenoid biosynthesis. Such phosphoantigens rapidly and selectively activate human gammadelta T cells to produce proinflammatory cytokines, notably interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). In addition, human gammadelta T cells express certain Toll-like receptors (TLR) and directly respond to the corresponding ligands. We have demonstrated expression of TLR3 in Vgamma9Vdelta2 T cells and striking costimulatory effects of the ligand polyinosinic-polycytidylic acid (polyI:C) on TCR-stimulated IFN-gamma production. Gene expression studies by microarray analysis identified additional genes that were up-regulated by combined TCR- and TLR3 stimulation. We discuss these findings in the context of the suspected role of human Vgamma9Vdelta2 T cells as a link between innate and adaptive immune responses.

Correlation of hemocyte counts with different developmental parameters during the last larval instar of the tobacco hornworm, Manduca sexta.

We determined the changes in hemocyte titer and in the abundance of hemocyte types of the tobacco hornworm Manduca sexta during the fourth and fifth larval stadium and the beginning of the pupal stadium. As we analyzed the samples of individual insects at daily intervals, we were able to correlate phenotypical features, body weight, as well as total protein content and lysozyme activity in the hemolymph with the observations on hemocytes. In the course of the fifth larval stadium, the hemocyte titer decreased slightly and declined further after pupation. Using calculated values for total hemocyte numbers, females had about five times and males three times more hemocytes in the circulating population at the beginning of the wandering stage (in the middle of the fifth larval stadium) than immediately after the last larval--larval molt (from the fourth to the fifth larval stadium). This sexual difference was mainly due to an increase in the number of plasmatocytes, which was more prominent in females than in males. Granular cells were dominant in early fifth larval stadium while plasmatocytes were the most abundant cells in pupae. Oenocytoids and spherule cells disappeared during the wandering stage. Lysozyme activity in the hemolymph rose to a maximum during the wandering stage, with females having lysozyme values twice as high as those for males. These changes in lysozyme activity, however, did not correlate with the increase of total hemolymph protein titer which occurred already at the beginning of the wandering stage. We postulate that changes in hemocyte titers are under direct hormonal control, which has to be proven in future experiments.

Characterization of terminal deoxynucleotidyl transferase and polymerase mu in zebrafish.

Terminal deoxynucleotidyl transferase (TdT) contributes to the junctional diversity of immunoglobulin and T-cell receptors by incorporating nucleotides in a template-independent manner. A closely related enzyme, polymerase mu (polmu), a template-directed polymerase, plays a role in general end-joining double-strand break repair. We cloned zebrafish TdT and polmu and found them to be 43% identical in amino acid sequence. Comparisons with sequences of other species revealed conserved residues typical for TdT in the zebrafish sequence that support the template independence of this enzyme. Some but not all of these features were identified in zebrafish polmu. In adult fish, TdT expression was most prominent in thymus, pro- and mesonephros, the primary lymphoid organs in teleost fish and in spleen, intestine, and the tissue around the intestine. Polmu expression was detected not only in pro- and mesonephros, the major sites for B-lymphocyte development, but also in ovary and testis and in all tissue preparations to a low extent. TdT expression starts at 4 dpf and increases thereafter. Polmu is expressed at all times to a similar extent. In situ studies showed a strong expression of TdT and polmicro in the thymic cortex of 8-week-old fish. The characterization of zebrafish TdT and polmu provide new insights in fish lymphopoiesis and addresses the importance and evolution of TdT and polmu themselves.

Human gamma delta T cells: candidates for the development of immunotherapeutic strategies.

A numerically small subset of human T lymphocytes expresses a gamma delta T cell receptor (TCR). These gamma delta T cells share certain effector functions with alpha beta T cells as well as with NK cells and NKT cells. The major peripheral blood gamma delta T cell subset in healthy adults expresses a Vgamma9Vdelta2 TCR, which recognizes small phosphorylated metabolites referred to as phosphoantigens. Vdelta1 gamma delta T cells mainly occur in the intestine. They recognize the stress-induced MICA/B and CD1c. Furthermore, gamma delta T cells express a variety of NK cell and pattern-recognition receptors which are responsible for the "fine-tuning" of effector functions. In recent years, gamma delta T cells start to emerge as a rewarding target for immunotherapeutic strategies against viral infections and cancer. A better understanding of factors that modulate gamma gamma delta T cell function will further eluminate the potential of these cells.

Direct costimulatory effect of TLR3 ligand poly(I:C) on human gamma delta T lymphocytes.

TLR3 recognizes viral dsRNA and its synthetic mimetic polyinosinic-polycytidylic acid (poly(I:C)). TLR3 expression is commonly considered to be restricted to dendritic cells, NK cells, and fibroblasts. In this study we report that human gammadelta and alphabeta T lymphocytes also express TLR3, as shown by quantitative real-time PCR, flow cytometry, and confocal microscopy. Although T cells did not respond directly to poly(I:C), we observed a dramatic increase in IFN-gamma secretion and an up-regulation of CD69 when freshly isolated gammadelta T cells were stimulated via TCR in the presence of poly(I:C) without APC. IFN-gamma secretion was partially inhibited by anti-TLR3 Abs. In contrast, poly(I:C) did not costimulate IFN-gamma secretion by alphabeta T cells. These results indicate that TLR3 signaling is differentially regulated in TCR-stimulated gammadelta and alphabeta T cells, suggesting an early activation of gammadelta T cells in antiviral immunity.

Differences between larval and pupal hemocytes of the tobacco hornworm, Manduca sexta, determined by monoclonal antibodies and density centrifugation.

Insect hemocytes play a major role in developmental processes where they disassociate and rebuild metamorphosing tissues while undergoing physiological changes themselves. We identified hemocyte changes from the last larval to the beginning of the pupal stage of the tobacco hornworm, Manduca sexta. Larval and pupal hemocytes behaved differently in a 40% Percoll density gradient. Larval granular cells were found in almost all density layers, pupal granular cells were abundant in high density layers; larval plasmatocytes occurred in dense layers, pupal plasmatocytes became enriched in less dense layers of the gradient. Using a panel of monoclonal antibodies generated against purified hemocytes, several different antibody binding patterns were identified. Quantitative differences in staining intensities were observed more often than qualitative changes, e.g. a loss or a gain of staining. Both phenomena were related to both plasmatocytes and granular cells. The distribution of the corresponding antigens in tissues was tested on cross sections of larvae and pupae as well as in Western blot analyses using organ homogenates. Several antibodies were specific for hemocytes only, among which two antibodies bound to molecules of the hematopoietic organ. Other antibodies had an additional reactivity to other tissues, mainly to the basal lamina.