CMV-Reactive Gamma/Delta T Cells Expanded From CMV Seronegative Normal Healthy Individuals Lyse CMV-Infected Targets – Potential Adoptive Immunotherapy for Recipients of Stem Cell Transplants From CMV Seronegative Donors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2233-2233
Author(s):  
Andrea Knight ◽  
Stephen Mackinnon ◽  
Mark W Lowdell
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cell Lines ◽  
Gamma Delta T Cells ◽  
Feeder Cells ◽  
Gamma Delta ◽  
T Cell Lines ◽  
Gamma Delta T Cell ◽  
Cmv Reactivation

Abstract Abstract 2233 Poster Board II-210 Reactivation of cytomegalovirus (CMV) early post-transplant remains a serious complication in lymphopenic recipients after allogeneic stem cell transplantation (SCT). CMV-specific alpha/beta T cells are the principal effector T cells in CMV infection in normal immunocompetent individuals. The role of gamma/delta T cells in anti-CMV responses is not currently fully understood. The majority of human circulating gamma/delta T cells express a TCR encoded by the Vgamma9 and Vdelta2 gene segments. Minor subset of circulating gamma/delta T cells known as Vdelta2neg cells (mainly Vdelta1 and Vdelta3) are expanded in CMV seropositive normal individuals compared to CMV seronegative donors (p=0.0002). CMV-reactive Vdelta2neg gamma/delta T cell lines were generated from seropositive healthy donors and shown to lyse CMV infected target cells in vitro. We have reported an expansion of the same subset of gamma/delta T cells in recipients of allogeneic SCT associated with CMV reactivation. Vdelta2neg cell lines were generated from a CMV+ patient who received a graft from a CMV+ sibling donor at two separate early time points post-transplant and also shown to be CMV-reactive with the ability to lyse CMV infected targets. Since gamma/delta T cells are part of the innate immune response we investigated whether CMV-reactive gamma/delta T cells can be isolated from CMV seronegative donors. Polyclonal Vdelta2neg gamma/delta T cell lines were raised and expanded from multiple CMV seronegative healthy subjects using feeder cells from seronegative and seropositive donors. Briefly, Vdelta2neg gamma/delta T cells were purified from peripheral blood mononuclear cells from CMV seronegative donors by immunomagnetic sorting and cultured in the presence of irradiated PBMC from two HLA-mismatched normal donors. Gamma/delta T cells were cultured for up to 5 weeks. The degree of gamma/delta T cell expansion ranged from 78-3250 fold and the purity of Vdelta2neg gamma/delta T cell lines averaged 88.3 %. Lines were co-cultured with uninfected MRC5 fibroblasts and CMV infected fibroblasts with TB40E or VHLE clinical CMV strains at different effector/target ratios. Vdelta2neg gamma/delta T cells showed specific cytotoxicity by lysis of CMV infected but not uninfected fibroblasts. The frequency of generation of CMV-reactive Vdelta2neg gamma/delta T cells was not affected by the use of feeder cells from seropositive versus seronegative donors. These results suggest that CMV-reactive Vdelta2neg gamma/delta T cells represent a novel approach for an adoptive immunotherapy for CMV seropositive recipients of grafts from CMV seronegative donors who are lacking protective CMV immunity and are at greater risk of CMV reactivation. CMV-reactive Vdelta2neg gamma/delta T cells generated from CMV seropositive donors could be used adoptively in patients suffering CMV reactivation without a risk of GvHD. Disclosures: Lowdell: Cell Medica Ltd: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Structure and specificity of T cell receptor gamma/delta on major histocompatibility complex antigen-specific CD3+, CD4-, CD8- T lymphocytes.

1988 ◽  
Vol 168 (5) ◽  
pp. 1899-1916 ◽  
Author(s):  
J A Bluestone ◽  
R Q Cron ◽  
M Cotterman ◽  
B A Houlden ◽  
L A Matis
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Gene Segment ◽  
Cell Receptor ◽  
Gamma Delta ◽  
Human T Cells ◽  
T Cell Lines ◽  
Gamma Delta T Cell ◽  
Delta Cells

Analyses of TCR-bearing murine and human T cells have defined a unique subpopulation of T cells that express the TCR-gamma/delta proteins. The specificity of TCR-gamma/delta T cells and their role in the immune response have not yet been elucidated. Here we examine alloreactive TCR-gamma/delta T cell lines and clones that recognize MHC-encoded antigens. A BALB/c nu/nu (H-2d)-derived H-2k specific T cell line and derived clones were both cytolytic and released lymphokines after recognition of a non-classical H-2 antigen encoded in the TL region of the MHC. These cells expressed the V gamma 2/C gamma 1 protein in association with a TCR-delta gene product encoded by a Va gene segment rearranged to two D delta and one J delta variable elements. A second MHC-specific B10 nu/nu (H-2b) TCR-gamma/delta T cell line appeared to recognize a classical H-2D-encoded MHC molecule and expressed a distinct V gamma/C gamma 4-encoded protein. These data suggest that many TCR-gamma/delta-expressing T cells may recognize MHC-linked antigens encoded within distinct subregions of the MHC. The role of MHC-specific TCR-gamma/delta cells in immune responses and their immunological significance are discussed.

scholarly journals Human fetal liver gamma/delta T cells predominantly use unusual rearrangements of the T cell receptor delta and gamma loci expressed on both CD4+CD8- and CD4-CD8- gamma/delta T cells.

1993 ◽  
Vol 177 (2) ◽  
pp. 425-432 ◽  
Author(s):  
K W Wucherpfennig ◽  
Y J Liao ◽  
M Prendergast ◽  
J Prendergast ◽  
D A Hafler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fetal Liver ◽  
Cell Populations ◽  
Gamma Delta T Cells ◽  
Gamma Delta ◽  
Gamma Chain ◽  
T Cell Clones ◽  
Cell Clones ◽  
Gamma Delta T Cell

Substantial numbers of both alpha/beta and gamma/delta T cells are present in human fetal liver, which suggests a role of the fetal liver in T cell development. The diversity of fetal liver T cell receptor (TCR) gamma and delta chain rearrangements was examined among both CD4+CD8- and CD4-CD8- gamma/delta T cell clones. In addition, TCR delta chain transcripts from three fetal livers were sequenced after polymerase chain reaction amplification of TCR delta chains with V delta 1 or V delta 2 rearrangements. Five of six fetal liver gamma/delta T cell clones had a V delta 2-D delta 3-J delta 3 gene rearrangement with limited junctional diversity; three of these clones had an unusual CD4+CD8- phenotype. V delta 2-D delta 3-J delta 3 gene rearrangements were also common among both in-frame and out-of-frame transcripts from three fetal livers, indicating that they are the result of an ordered rearrangement process. TCR gamma chain sequences of the fetal liver gamma/delta T cell clones revealed V gamma 1-J gamma 2.3, V gamma 2-J gamma 1.2, and V gamma 3-J gamma 1.1 rearrangements with minimal incorporation of template-independent N region nucleotides. TCR gamma chain rearrangements found in these fetal liver T cell clones were different from those that have been observed among early thymic gamma/delta T cell populations, while similar TCR delta chain rearrangements are found among gamma/delta T cells from both sites. These data demonstrate that the fetal liver harbors gamma/delta T cell populations distinct from those found in the fetal thymus, suggesting that the fetal liver is a site of gamma/delta T cell development in humans. These unusual T cell populations may serve a specific function in the fetal immune system.

scholarly journals Intercellular interactions and cytokine responsiveness of peritoneal alpha/beta and gamma/delta T cells from Listeria-infected mice: synergistic effects of interleukin 1 and 7 on gamma/delta T cells.

1993 ◽  
Vol 178 (3) ◽  
pp. 985-996 ◽  
Author(s):  
M J Skeen ◽  
H K Ziegler
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
T Cell Subsets ◽  
Gamma Delta T Cells ◽  
Gamma Delta ◽  
Accessory Cells ◽  
Alpha Beta ◽  
Gamma Delta T Cell

Peritoneal gamma/delta T cells from Listeria-immune mice show an enhanced potential to expand when restimulated with antigens or mitogens in vitro (see companion paper [Skeen, M. J., and H. K. Ziegler. 1993. J. Exp. Med. 178:971]). When cocultured with peritoneal alpha/beta T cells, the gamma/delta T cell population expanded preferentially even when the in vitro stimulus was specific for the alpha/beta T cell population. Purified gamma/delta T cells did not respond to alpha/beta T cell-specific stimuli. If isolated T cell subsets were recombined in cell mixing experiments, the resulting proliferative response was greater than additive. Irradiated alpha/beta T cells could enhance the proliferation of responding gamma/delta T cells, but the effect was unidirectional; i.e., irradiated gamma/delta T cells did not stimulate responding gamma/delta T cells. This effect appeared to be cytokine mediated and did not require cell-cell contact. Both recombinant interleukin 2 (rIL-2) and rIL-7 could support the expansion of the gamma/delta T cells, while rIL-7 was only minimally stimulatory for the alpha/beta T cells. The magnitude of the response by gamma/delta T cells to rIL-7 exceeded the response to other in vitro stimuli, including immobilized anti-T cell receptor monoclonal antibody, and was 50-100-fold greater than the alpha/beta T cell response to IL-7. This unique sensitivity of gamma/delta T cells to IL-7 was strongly enhanced by the presence of accessory cells. These cells could be replaced by rIL-1, establishing a synergy for IL-1 and IL-7 as factors that could uniquely stimulate this gamma/delta T cell population. Isolated peritoneal gamma/delta T cells from Listeria-immune mice react to heat-killed Listeria preparations in the presence of macrophages accessory cells in a non-H-2-restricted manner. Considered collectively, these results suggest a potential mechanism by which gamma/delta T cells can predominate in epithelial tissues and at sites of infection.

scholarly journals Induction of murine peritoneal gamma/delta T cells and their role in resistance to bacterial infection.

1993 ◽  
Vol 178 (3) ◽  
pp. 971-984 ◽  
Author(s):  
M J Skeen ◽  
H K Ziegler
Keyword(s):  
T Cells ◽  
T Cell ◽  
Peritoneal Cavity ◽  
Gamma Delta T Cells ◽  
Gamma Delta ◽  
Gram Positive ◽  
Gram Negative ◽  
A Site ◽  
Gamma Delta T Cell

Previous studies have reported an association of gamma/delta T cells with microbial infection in both human lesions and murine infectious disease models. In this study we provide a comprehensive analysis of the conditions under which the induction of gamma/delta T cells occurs at a site of infection. We found a site-specific induction of gamma/delta T cells after the injection of Listeria monocytogenes in the peritoneal cavity of C3H mice. No changes were seen in the splenic or lymph node populations after these injections. Both the proportion and the absolute number of gamma/delta T cells increased in the peritoneal cavity. Additionally, when peritoneal T cells from Listeria-immune mice were restimulated in vitro, the induced gamma/delta T cells exhibited a greater expansion potential than the alpha/beta T cells. Neither the induced gamma/delta T cells nor those from normal mice expressed CD4 or CD8 on the cell surface. Thy-1 was expressed on only 29% of normal peritoneal gamma/delta T cells, but after intraperitoneal Listeria injection 65% of induced gamma/delta T cells expressed. Thy-1, Pgp-1 and CD45R expression on both normal and induced gamma/delta T cells was consistent with an activation phenotype. Significant increases in peritoneal gamma/delta T cells were not seen until 5-7 d after Listeria injection. The proportion of the CD3+ population expressing the gamma/delta T cell receptor remained elevated for 6-7 wk, while the absolute numbers of peritoneal gamma/delta T cells declined gradually over this time period, reflecting a decrease in both the number of lymphocytes and the percentage of these that were CD3+. Peak numbers of gamma/delta T cells were seen at day 10 with live microbes such as Listeria. A variety of microbes, toxins, mitogens, antigens, cytokines, and nonspecific inflammatory agents were evaluated for their ability to induce gamma/delta T cells in the peritoneal cavity. Both Gram-positive and Gram-negative bacteria as well as Mycobacteria were able to induce gamma/delta T cells that showed increased in vitro expansion potential. An exotoxin from a Gram-positive organism, listeriolysin-o, and the lipopolysaccharide (LPS) endotoxin from a Gram-negative organism were also effective. gamma/delta T cell responses to LPS were under lps gene control. Peak numbers of gamma/delta T cells were observed at day 3 after injection with exotoxins and endotoxins. Modifications that abrogated the virulence of a bacterial strain also eliminated the inductive effect for gamma/delta T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid generation of combined CMV-specific CD4+ and CD8+ T-cell lines for adoptive transfer into recipients of allogeneic stem cell transplants

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3565-3572 ◽  
Author(s):  
Georg Rauser ◽  
Hermann Einsele ◽  
Christian Sinzger ◽  
Dorothee Wernet ◽  
Gabriele Kuntz ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cell Lines ◽  
Adoptive Transfer ◽  
Cd8 T Cell ◽  
Cell Transplants ◽  
Ifn Γ ◽  
Rapid Generation ◽  
T Cell Lines

Abstract Adoptive transfer of cytomegalovirus (CMV)-specific T cells can restore long-lasting, virus-specific immunity and clear CMV viremia in recipients of allogeneic stem cell transplants if CD4+ and CD8+ CMV-specific T cells are detected in the recipient after transfer. Current protocols for generating virus-specific T cells use live virus, require leukapheresis of the donor, and are time consuming. To circumvent these limitations, a clinical-scale protocol was developed to generate CMV-specific T cells by using autologous cellular and serum components derived from a single 500-mL blood draw. CMV-specific T cells were stimulated simultaneously with CMV-specific major histocompatibility complex class I (MHC I)- restricted peptides and CMV antigen. Activated T cells were isolated with the interferon-γ (IFN-γ) secretion assay and expanded for 10 days. In 8 randomly selected, CMV-seropositive donors, 1.34 × 108 combined CD4+ and CD8+ CMV-specific T cells, on average, were generated, as determined by antigen-triggered IFN-γ production. CMV-infected fibroblasts were efficiently lysed by the generated T cells, and CMV-specific CD4+ and CD8+ T cells expanded if they were stimulated with natural processed antigen. On the other hand, CD4+ and CD8+ T cell-mediated alloreactivity of generated CMV-specific T-cell lines was reduced compared with that of the starting population. In conclusion, the culture system developed allowed the rapid generation of allodepleted, highly enriched, combined CD4+ and CD8+ CMV-specific T cells under conditions mimicking good manufacturing practice. (Blood. 2004; 103:3565-3572)

scholarly journals Zoledronic Acid Induces the Proliferation of Human Cord Blood Gamma Delta T Cells Ex Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1427-1427
Author(s):  
Suzanne L Tomchuck ◽  
Jin He ◽  
Ross W. Perko ◽  
Scarlett Evans ◽  
Amy McKenna ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Fold Increase ◽  
T Cell Proliferation ◽  
Gamma Delta T Cells ◽  
Memory Cells ◽  
Gamma Delta ◽  
Gamma Delta T Cell

Abstract Cord blood (CB) T cells are known to be naïve cells, but can be activated to respond similar to adult peripheral blood (PB) T cells. Reports indicate that culture with aminobisphosphonate (NBP) stimulates CB gamma delta T cell proliferation ex vivo, specifically the TCRγ9δ2 subset, which has been extensively studied in PB gamma delta T cells. As CB gamma delta T cells are not well described, we compared CB gamma delta T cell proliferation, phenotype and genotype to PB gamma delta T cells when culturing cells with the NBP, Zometa (zoledronic acid), and IL-2. Fourteen days in culture resulted in significant fold increase in the proliferation of gamma delta T cells and in the percent of lymphocytes in both sample types. PB gamma delta T cells proliferated more robustly than CB with a 288.60 versus 21.32 fold increase, respectively. Additionally, in freshly isolated samples, CB gamma delta T cells comprised an average of 1.404% of the lymphocyte population, which was similar to PB gamma delta T cells, with an average of 2.319%. However, by day 14, PB gamma delta T cells increased to 70.15% of lymphocytes whereas CB gamma delta T cells increased to 12.49%. Phenotypically, both CB and PB had similar percent of CD45RA+ and CD45RO+ gamma delta T cell memory subsets in freshly isolated samples. Following culture, PB gamma delta T cells were mostly CD45RO+ memory cells, with significantly fewer CD45RA+ naïve cells, whereas more CB gamma delta T cells were of the intermediate CD45RA+CD45RO+ subset. Further phenotypic analysis of the memory subsets indicated that cultured PB gamma delta T cells were either effector memory cells (CD27-CD45RA-) or central memory cells (CD27+CD45RA-), while CB gamma delta T cells were mostly naïve (CD27+CD45RA+). The cytokines secreted by these cells were also assessed and the culture of PB and CB gamma delta T cells resulted in differing cytokine secretion profiles. After 14 days of culture, PB gamma delta T cells secreted more IFNγ and TNFα, while CB gamma delta T cells secreted more IL-10 and RANTES. We also examined TCRγ9 and TCRδ2 phenotypic expression and found that the TCRγ9δ2 was a common clone in freshly isolated PB gamma delta T cells, which predominated after 14 days in culture. However, while the TCRγ9δ2 variant was expressed in CB gamma delta T cells, it was low before and after culture, suggesting that Zometa may not stimulate gamma delta T cells in CB the same as PB. As limited TCRγδ phenotypic reagents are available, we developed a single cell PCR assay for genotypic analysis of the TCRγδ repertoire. PCR analysis suggests that the TCRγδ repertoire is diverse in both samples, yet TCRγ9δ2 is most prevalent. Further analysis of the variant subsets is warranted and may give insight into how each of these receptor pairings affects function. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ontogenic development and tissue distribution of V gamma 1-expressing gamma/delta T lymphocytes in normal mice.

1995 ◽  
Vol 182 (6) ◽  
pp. 1921-1930 ◽  
Author(s):  
P Pereira ◽  
D Gerber ◽  
S Y Huang ◽  
S Tonegawa
Keyword(s):  
T Cells ◽  
T Cell ◽  
Intestinal Epithelium ◽  
Lymphoid Organs ◽  
Cell Subpopulation ◽  
Postnatal Life ◽  
Gamma Delta T Cells ◽  
Gamma Delta ◽  
Adult Mice ◽  
Gamma Delta T Cell

A hamster monoclonal antibody (mAb) recognizing an epitope in the V gamma 1-J gamma 4-C gamma 4 chain of the gamma/delta T cell receptor has been generated. Using this mAb, we have quantitated the occurrence of V gamma 1-bearing gamma/delta T cells in the developing thymus and in the lymphoid organs and several epithelia of adult mice. The V gamma 1-expressing cells constitute a minor gamma/delta T cell subpopulation during fetal and early postnatal life, but they constitute a major population of gamma/delta T cells in the thymus and in the peripheral lymphoid organs in adult mice. In addition, we found that V gamma 1-bearing cells comprise a large proportion (15-60%) of the gamma/delta T cells present in the intestinal epithelium (i-IEL) in all strains of mice tested. V gamma 1+ i-IEL are present in athymic (nude) mice and in antigen-free mice, demonstrating that they can develop extrathymically and that their presence in the intestinal epithelium is independent of the antigenic load of the gut. Our results show that V gamma 1-bearing lymphocytes account for the largest population of gamma/delta T cells in the mouse. This population includes a thymus-dependent component that homes to the secondary lymphoid organs and a thymus-independent component that constitutes a major fraction of the gamma/delta i-IELs.

scholarly journals Fetal liver T cell receptor gamma/delta+ T cells as cytotoxic T lymphocytes specific for maternal alloantigens.

1992 ◽  
Vol 176 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Y Miyagawa ◽  
T Matsuoka ◽  
A Baba ◽  
T Nakamura ◽  
T Tsuno ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Lines ◽  
Fetal Liver ◽  
Cell Receptor ◽  
Lymphoid Cells ◽  
Gamma Delta T Cells ◽  
Gamma Delta ◽  
Killer Activity

We have established fetal liver-derived T cell receptor (TCR) gamma/delta+, CD3+ T cell lines that are cytotoxic for maternal T cells. Fetal liver-derived lymphoid progenitors yielded predominantly TCR-gamma/delta+ cell clusters when cultured on fetal bone marrow-derived stromal cells in the presence of a cytokine cocktail under magnetic force. These tightly adherent clusters were cloned by limiting dilution and the resulting cell lines analyzed for phenotype and function. Six of eight TCR-gamma/delta lines from 8-9.5-wk gestation fetuses were V delta 2+ as compared with zero of eight lines from later stages of gestation (10 and 15 wk), where all the lines were V delta 1+. In cytotoxicity assays, these TCR-gamma/delta+, CD3+, CD4-, and CD8+ or CD8- long-term cultured lymphoid cells (LLC) were killer cells active against the class I antigens on maternal T cells. Of the cell lines, the CD8+ TCR-gamma/delta+ LLC had the highest levels of killer activity. Thus fetal liver TCR-gamma/delta+ T cells may play a crucial role in protection against invading maternal T cells generated in the feto-maternal interaction.

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