scholarly journals Blocking of EphA2 on Endometrial Tumor Cells Reduces Susceptibility to Vδ1 Gamma-Delta T-Cell-Mediated Killing

2021 ◽  
Vol 12 ◽  
Author(s):  
Robert Hudecek ◽  
Barbora Kohlova ◽  
Ingrid Siskova ◽  
Martin Piskacek ◽  
Andrea Knight
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Peritoneal Fluid ◽  
Γδ T Cells ◽  
Gamma Delta ◽  
Healthy Donors ◽  
Endometrial Tumor

BackgroundEndometriosis is a common gynecological disease characterized by the presence of endometrial tissue outside the uterus causing chronic inflammation, severe pain, and infertility. However, the innate immunity of gamma-delta (γδ) T lymphocytes in endometriosis has not been characterized. Women with endometriosis present numerous endocrine and immune dysfunctions and elevated risk for endometrial, ovarian, and breast cancers. The tyrosine kinase EphA2 is often overexpressed in cancer including endometrial carcinoma.MethodsWe analyzed Vδ1 and Vδ2 γδ T cells in peripheral blood and paired peritoneal fluid samples in endometriosis patients (n = 19) and compared the counts with that of age- and sex-matched healthy donors (n = 33) using flow cytometry. Vδ1 and Vδ2 T cells isolated from healthy donors were used against KLE, RL-95, and Ishikawa endometrial tumor cells in 4 h flow cytometric cytotoxicity assays. The EphA2 blocking studies were performed using antibody, small-molecule inhibitor ALW-II-41-27, and the CRISPR/Cas9.ResultsWe determined Vδ1 T cells substantially reduced in patients’ peripheral blood (p < 0.01) and peritoneal fluid (p < 0.001). No differences were found for circulating Vδ2 T cells compared with peritoneal fluid samples. We observed inherent cytotoxic reactivity of Vδ1 and Vδ2 γδ T lymphocytes against endometrial tumor cells. Importantly, we found reduced specific lysis of EphA2-positive cell lines KLE and RL-95 by Vδ1 T cells in the EphA2 antibody blocking studies and by the EphA2 inhibitor. Furthermore, Vδ1 T-cell-mediated killing was significantly decreased in RL-95 cell EPHA2 knockout. Finally, potent cytolytic activity exerted by Vδ1 T cells was significantly reduced in EPHA2 knockouts in renal A-498 and colon HT-29 carcinoma cell lines.ConclusionsWe determined variable levels of Vδ1 and Vδ2 γδ T cells in endometriosis patients. We observed inherent cytotoxic reactivity of γδ T-cell subsets against endometrial cell lines. Specifically, we found that blocking of EphA2 expression resulted in significant inhibition of endometrial tumor killing mediated by Vδ1 γδ T cells. These results suggest that EphA2 is involved in tumor cell lysis and contributes to susceptibility to Vδ1 γδ T cells cytotoxic reactivity.

scholarly journals 688 In vivo expansion of gamma delta T cells by a CD19-targeted butyrophilin heterodimer leads to elimination of peripheral B cells

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A727-A727
Author(s):  
Suresh De Silva ◽  
George Fromm ◽  
Louis Gonzalez ◽  
Arpita Patel ◽  
Kyung Yoon ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Mechanism Of Action ◽  
Peripheral Blood ◽  
Fusion Proteins ◽  
Gamma Delta T Cells ◽  
Gamma Delta ◽  
Gamma Delta T Cell

BackgroundA primary mechanism of cancer immunotherapy resistance involves downregulation of specific antigens or major histocompatibility complex based antigen presentation, which renders tumor cells invisible to alpha-beta T cells, but not gamma-delta T cells. Recently, a two-step model of gamma-delta T cell activation has emerged, wherein one butyrophilin (BTN, ie. BTN2A1) directly binds the gamma-delta TCR but is only activated if certain molecular patterns (eg. phosphoantigens) facilitate recruitment of a second BTN (ie. BTN3A1) into a complex to form a BTN2A1/3A1 heterodimer. The BTN2A1/3A1 complex specifically activates the predominant gamma-delta T cell population in the peripheral blood, comprising the Vg9d2 T cell receptor (TCR), but does not activate the primary gamma-delta T cell population in mucosal tissues, comprising the Vg4 TCR. The unique mechanism of action and specificity of gamma-delta TCR/BTN interactions suggests that therapeutic proteins comprising specific BTN heterodimers could be used to target specific gamma-delta T cell populations, with a lower risk of off-target activation common with CD3-directed T cell engagers.MethodsHuman BTN2A1/3A1-Fc-CD19scFv and mouse BTNL1/6-Fc-CD19scFv heterodimeric fusion proteins were purified and binding to CD19 or the respective gamma-delta TCRs was assessed by ELISA, Octet and flow cytometry using gd T-cells isolated from human peripheral blood and mouse intestinal tissue. The functionality of the constructs to activate gamma-delta T cells and mediate killing of tumor cells was assessed using live cell imaging in vitro as well as a murine B-cell lymphoma model in vivo.ResultsThe CD19-targeting scFv domains of the BTN heterodimer fusion proteins bound to human and mouse CD19 with low nanomolar affinity. The BTN2A1/3A1-Fc-CD19scFv compound specifically bound to the Vg9d2 TCR on human gd T cells while the mouse BTNL1/6-Fc-CD19scFv bound to Vg7d4 TCR on mouse gd T cells. Both compounds were able to activate gd T cells in a co-culture assay resulting in degranulation and increased surface expression of CD107a and also increased apoptosis of CD19+ tumor cells. Intraperitoneal administration of the mouse BTNL1/6-Fc-CD19scFv led to anti-tumor effects in A20 tumor bearing BALB/c mice. Phenotyping from BTNL1/6-Fc-CD19scFv treated mice revealed profound and rapid expansion of the endogenous gamma-delta T cells in the circulation and tumor, with concomitant depletion of peripheral CD19+ B-cells, confirming the mechanism of action of the heterodimer as a gamma-delta T cell specific engager.ConclusionsThese results provide proof of mechanism for in vivo manipulation of gamma-delta T cells using antigen-targeted butyrophilin heterodimeric fusion proteins for the treatment of cancer.

In Vitro Expansion of Human Tumor-Reactive CD8+ T Cell Lines Using Superparamagnetic CD3/CD28/CD137 Beads.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5118-5118
Author(s):  
Daniel Teschner ◽  
Eva Distler ◽  
Elke Schnuerer ◽  
Gregor Wenzel ◽  
Axl Neurauter ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Cd8 T Cell ◽  
Strong Increase ◽  
In Vitro Expansion ◽  
T Cell Lines

Abstract Abstract 5118 Introduction Efficient methods for the reliable in vitro expansion of tumor-reactive T cells will surely broaden the applicability of adoptive T cell therapy in cancer. In this study we investigated the antigen-independent stimulation and expansion of human T cells in peripheral blood mononuclear cells (PBMC) and in long-term cultured tumor-reactive CD8+ T cell lines using superparamagnetic beads coated with antibodies to CD3 and the costimulatory molecules CD28 and CD137. Methods T cell numbers were measured in healthy donor PBMC after in vitro stimulation with Dynabeads® coated with CD3/CD28/CD137 versus Dynabeads® coated with CD3/CD28 (all beads +/- 100 U/mL IL-2) versus IL-2 alone at different bead/cell ratios (3:1, 1:1). Expansion was also analyzed in human renal cell carcinoma-reactive CD8+ T cell lines after restimulation with tumor cells (weekly), CD3/CD28 beads and CD3/CD28/CD137 beads, respectively (bead/cell ratio of 1:5, 100 U/mL IL-2 added). Expanded T cell lines were phenotyped for expression of activation, differentiation and homing molecules (i.e. CD27, CD28, CD45RA, CD45RO, CD57, CD62L, CD137, CCR7) and were also tested for function. Results T cells in PBMC showed an increased expansion rate of up to 17-fold during a 2-week culture period using beads with IL-2 added versus IL-2 alone (p<0.0001 for CD3/CD28/CD137; p<0.0001 for CD3/CD28). The difference between CD3/CD28/CD137 beads and CD3/CD28 beads was not significant (p=0.4). Bead/cell ratios of 1:1 and 3:1 expanded T cells in PBMC with similar efficiency. In addition, IL-2 was essential to obtain maximum T cell proliferation. Peripheral blood CD4+ and CD8+ T cells showed a strong increase of CD137 surface expression starting 12-24 hours upon stimulation, regardless which beads were used. In contrast to PBMC, tumor-reactive CD8+ T cell lines expanded more rapidly using CD3/CD28/CD137 beads versus CD3/CD28 beads (p=0.03). Stimulation with CD3/CD28/CD137 beads was comparably efficient versus the control arm using weekly addition of tumor cells and IL-2. Simultaneous addition of beads and tumor cells did not have a synergistic effect. CD8+ T cell lines analyzed 12 days after bead-induced in vitro expansion versus weekly tumor stimulation showed a comparable level of tumor reactivity in IFN-g ELISPOT assay. Phenotypically, expression of CD137 on CD8+ T cell lines showed maximum up-regulation 24 hours after beads stimulation and persisted for at least 72 hours. In contrast, cultures stimulated solely with tumor cells showed a much shorter and transient CD137 expression with an earlier peak level after 12 hours. Other phenotypic markers were similar on tumor-reactive T cell cultures, except for increased CD62L expression after bead-induced stimulation. Conclusion Antigen-independent in vitro expansion of T cells in PBMC was equally efficient using CD3/CD28 beads or CD3/CD28/CD137 beads, respectively. In contrast, we observed an increased growth rate for tumor-reactive CD8+ T cell lines when activated with CD3/CD28/CD137 beads compared to CD3/CD28 beads. Antitumor reactivity of T cell lines was maintained during the antigen-independent stimulation step. Bead activation was associated with increased expression of the lymph node homing receptor CD62L on antitumor CD8+ T cell lines, which indicates a central memory phenotype. Our data suggest that the conjugation of anti-CD137 antibodies to the traditionally used CD3/CD28 beads improves their expansion capacity for antitumor CD8+ T cell lines. Disclosures No relevant conflicts of interest to declare.

scholarly journals Profile of gamma-delta (γδ) T lymphocytes in the peripheral blood of crossbreed dogs during stages of life and implication in aging

2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Cristina Marchetti ◽  
Paolo Borghetti ◽  
Antonio Cacchioli ◽  
Luca Ferrari ◽  
Federico Armando ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Γδ T Cells ◽  
Γδ T Cell ◽  
Gamma Delta ◽  
Intact Male ◽  
Age Range ◽  
First Time

Abstract Background Data on gamma-delta (γδ) T lymphocytes in the peripheral blood of dogs are scant, related only to healthy pure breed dogs and limited to a restricted age range. The aim of the study was to investigate the modulation of the γδ T lymphocyte (TCRγδ+) subpopulation in peripheral blood of crossbreed healthy dogs according to five identified stages of life: Puppy, Junior, Adult, Mature, Senior and to determine its implication in aging. A rigorous method of recruitment was used to minimize the influence of internal or external pressure on the immune response. Twenty-three intact female and twenty-four intact male dogs were enrolled. Blood samples were collected and immunophenotyping of peripheral blood T lymphocytes and γδ T cell subpopulations was performed. Results The percentage of γδ T cells in peripheral blood lymphocytes was comparable with the value of 2.5% published by Faldyna and co-workers (2001), despite the percentage reported was investigated in less arranged age range groups and coming from four different dog pure breeds, whereas our data were recorded on wider age range groups and coming from crossbreed dogs. Therefore, the γδ T cell percentage (2.5%) is consistent and points out that such value is breed-independent. Statistical analysis highlighted differences in both percentage and absolute γδ T cells according to the stage of life. γδ T cells decreased significantly in the peripheral blood of elder dogs (Senior group) in comparison with previous stages of life (Puppy, Junior, and Adult groups). Differences in γδ T cells are significant and they are reported, for the first time, related to dog aging. Conclusions The study confirms dogs to be among the animals with a low TCRγδ+ cell profile. A decrease of the TCRγδ+ subpopulation percentage was observed in elder dogs. TCRγδ+ cells of group S were different from those of groups P, J, and A. The differences are reported for the first time in dog aging. Identifying the stage of life when the decrease of γδ T lymphocytes starts can be useful for providing a rationale for drafting a wellness plan trial to support thymus immune functions and mitigate its functional exhaustion.

scholarly journals Bovine γδ T-Cell Responses to the Intracellular Protozoan Parasite Theileria parva

1999 ◽  
Vol 67 (5) ◽  
pp. 2241-2249 ◽  
Author(s):  
Claudia A. Daubenberger ◽  
Evans L. N. Taracha ◽  
Laima Gaidulis ◽  
William C. Davis ◽  
Declan J. McKeever
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Γδ T Cells ◽  
Theileria Parva ◽  
Γδ T Cell ◽  
Mhc Molecules ◽  
T Cell Lines

ABSTRACT T cells bearing the γδ antigen receptor (γδ T cells) can constitute up to 50% of T cells in the peripheral blood and lymphoid organs of young cattle. We present data showing that γδ T cells are involved in immune responses against Theileria parva. γδ T cells isolated from peripheral blood mononuclear cells (PBMC) of T. parva-naive and -immune cattle proliferated in the presence of fixed or unfixed autologous T. parva-infected lymphoblasts (TpL) and heat-stressed concanavalin A (ConA)-induced blasts (ConA blasts) but not untreated ConA blasts. The specificity of response was further evaluated with a panel of γδ T-cell lines and clones. T-cell reactivity was blocked by GB21A, a monoclonal antibody (MAb) specific for the γδ T-cell receptor, but not by MAbs specific for class I and class II major histocompatibility complex (MHC) molecules. In addition, TpL but not ConA blasts from a variety of MHC-mismatched animals induced proliferation of the γδ T-cell lines and clones. These γδ T cells were found to respond to TpL infected with several different parasite stocks and failed to recognize TpL after elimination of the parasite by the theilericidal drug BW 720C. Assays for cytotoxic activity of γδ T cells sorted from bulk cultures of immune PBMC restimulated several times with autologous TpL demonstrated that effector cells whose specificity is similar to that of proliferating cells are generated. These results suggest that bovine γδ T cells are activated by and lyse T. parva-infected cells by recognizing conserved parasite-induced or parasite-derived antigens in an MHC-unrestricted fashion.

scholarly journals Regulatory T Cells Fail to Suppress Fast Homeostatic Proliferation In Vitro

Life ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 245
Author(s):  
Daniil Shevyrev ◽  
Valeriy Tereshchenko ◽  
Elena Blinova ◽  
Nadezda Knauer ◽  
Ekaterina Pashkina ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Autoimmune Diseases ◽  
Peripheral Blood ◽  
Treg Cells ◽  
Homeostatic Proliferation ◽  
Suppressive Activity ◽  
Healthy Donors

Homeostatic proliferation (HP) is a physiological process that reconstitutes the T cell pool after lymphopenia involving Interleukin-7 and 15 (IL-7 and IL-15), which are the key cytokines regulating the process. However, there is no evidence that these cytokines influence the function of regulatory T cells (Tregs). Since lymphopenia often accompanies autoimmune diseases, we decided to study the functional activity of Tregs stimulated by HP cytokines from patients with rheumatoid arthritis as compared with that of those from healthy donors. Since T cell receptor (TCR) signal strength determines the intensity of HP, we imitated slow HP using IL-7 or IL-15 and fast HP using a combination of IL-7 or IL-15 with anti-CD3 antibodies, cultivating Treg cells with peripheral blood mononuclear cells (PBMCs) at a 1:1 ratio. We used peripheral blood from 14 patients with rheumatoid arthritis and 18 healthy volunteers. We also used anti-CD3 and anti-CD3 + IL-2 stimulation as controls. The suppressive activity of Treg cells was evaluated in each case by the inhibition of the proliferation of CD4+ and CD8+ cells. The phenotype and proliferation of purified CD3+CD4+CD25+CD127lo cells were assessed by flow cytometry. The suppressive activity of the total pool of Tregs did not differ between the rheumatoid arthritis and healthy donors; however, it significantly decreased in conditions close to fast HP when the influence of HP cytokines was accompanied by anti-CD3 stimulation. The Treg proliferation caused by HP cytokines was lower in the rheumatoid arthritis (RA) patients than in the healthy individuals. The revealed decrease in Treg suppressive activity could impact the TCR landscape during lymphopenia and lead to the proliferation of potentially self-reactive T cell clones that are able to receive relatively strong TCR signals. This may be another explanation as to why lymphopenia is associated with the development of autoimmune diseases. The revealed decrease in Treg proliferation under IL-7 and IL-15 exposure can lead to a delay in Treg pool reconstitution in patients with rheumatoid arthritis in the case of lymphopenia.

TIGIT Induces (CD3+) T Cell Dysfunction by Inhibiting Glucose Metabolism and its Reversal by TIGIT and/or PD-1 Blockade in Colorectal Cancer

2021 ◽  
Author(s):  
qi shao ◽  
Lei Wang ◽  
maoling yuan ◽  
Xiaohong Jin ◽  
changping wu
Keyword(s):  
Colorectal Cancer ◽  
T Cells ◽  
T Cell ◽  
Glucose Metabolism ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Peripheral Blood ◽  
Cancer Tissue ◽  
Cell Dysfunction ◽  
T Cell Dysfunction

Abstract Background: T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is an immunosuppressive receptor expressed on the surface of immune cells, suppressing immune responses by activating the intracellular negative regulatory signals. TIGIT plays an important role in the pathogenesis of various tumors, but its immune escape in colorectal cancer remains unclear.Methods: In this study, TIGIT expression in the peripheral blood and tissue microarrays was detected flow cytometry and immunofluorescence and its relationship with prognosis was evaluated. The proliferation and cytokines of TIGIT+ T cells were measured. Glucose metabolism and key enzymes were detected by qPCR or western blot. After establishing the co-cultured system and xenotransplant models, TIGIT antibody alone or combined with PD-1 antibody was blocked to observe the tumor growth.Results: We found that the proportion of CD3+TIGIT+ T cells was increased in peripheral blood and cancer tissue in colorectal cancer patients when compared with the healthy donors. These cells exhibited functional defects, low proliferative activity, impaired cytokine production and reduced glucose metabolism. A strong association was also observed between the elevated TIGIT expression and poor prognosis. In the in vitro co-culture assays of T cells and tumor cells, the suppressed glucose metabolic activity of T cells was reversed by TIGIT blockade. In addition, this blockade induced the apoptosis and reduced G2/M transit in tumor cells. The antitumor efficacy of TIGIT Ab therapy was further demonstrated in a human colorectal xenograft mice model while co-blockers of TIGIT and PD-1 exhibited synergistic suppressing effects on tumor growth.Conclusions: It is suggest that while TIGIT induces CD3+ T cell dysfunction in colorectal cancer, co-targeting TIGIT and PD-1 can lead to an effective antitumor response and may serve as a novel therapeutic strategy for colorectal patients.

scholarly journals Cytoplasmic expression of the CD3 antigen as a diagnostic marker for immature T-cell malignancies

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 603-612
Author(s):  
JJ van Dongen ◽  
GW Krissansen ◽  
IL Wolvers-Tettero ◽  
WM Comans-Bitter ◽  
HJ Adriaansen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Diagnostic Marker ◽  
Surface Membrane ◽  
Cell Lineage ◽  
Normal Peripheral Blood ◽  
Thymus Cell ◽  
T Cell Malignancies

The expression of cytoplasmic CD3 (CyCD3) was analyzed in 45 leukemias, five thymus cell samples, five peripheral blood (PB) samples, and ten cell lines. All T cell acute lymphoblastic leukemias (T-ALL) that did not express surface membrane CD3 (SmCD3) appeared to express CyCD3. Furthermore, the majority of SmCD3+ T-ALL also expressed CyCD3. Analogous results were obtained with thymus cell samples in that about 95% of the thymocytes expressed CyCD3 whereas 60% to 75% of the thymocytes also expressed SmCD3. In normal peripheral blood only prominent SmCD3 expression was found. These data indicate that immature T cells express CyCD3 only, that the combined expression of CyCD3 and SmCD3 is characteristic for intermediate differentiation stages, and that mature T cells express prominent SmCD3. All (precursor) B cell leukemias, acute myeloid leukemias, and non-T cell lines tested did not express CyCD3. On the basis of these data, we conclude that CyCD3 expression is restricted to the T cell lineage and can be used as a diagnostic marker for immature SmCD3- T cell malignancies. Therefore, we evaluated which fixative is optimal for CyCD3 staining, and we determined by immunofluorescence staining and Western blotting which anti-CD3 monoclonal antibody (MoAb) can be used for the detection of CyCD3. In our opinion, acid ethanol was the best fixative for the cytocentrifuge preparations. Furthermore, we demonstrated that CyCD3 can be easily detected by use of MoAbs raised against denaturated CD3 chains such as those of the SP series (SP-6, SP-10, SP-64, and SP-78). In addition we tested 22 anti-CD3 MoAbs of the Oxford CD3 panel that were raised against native SmCD3, and it appeared that only four (UCHT1, VIT-3b, G19–41 and SK7/Leu-4) of them were able to detect CyCD3. In Western blot analysis all four MoAbs recognized the CD3- epsilon chain only.

scholarly journals T-cell-subset characterization of human T-CLL

Blood ◽  
1979 ◽  
Vol 53 (6) ◽  
pp. 1066-1075 ◽  
Author(s):  
EL Reinherz ◽  
LM Nadler ◽  
DS Rosenthal ◽  
WC Moloney ◽  
SF Schlossman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Peripheral Blood ◽  
Lymphoblastic Leukemia ◽  
Cell Subset ◽  
T Cell Subsets ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Human T Cell ◽  
Malignant T Cells

Abstract Circulating peripheral blood tumor cells in four cases of chronic lymphoproliferative disease were immunologically characterized. By the use of T-cell-specific heteroantisera and indirect immunofluorescence, all were shown to involve proliferation of malignant T cells. Three cases demonstrated morphologic and clinical features consistent with chronic lymphocytic leukemia (CLL), and one case presented as a lymphosarcoma cell leukemia. Antisera specific for normal human T-cell subsets defined the malignant T cells in each case as arising from the TH2--subset. This subset normally constitutes approximately 80% of human peripheral blood T cells. Terminal deoxynucleotidyl transferase (TdT) was not detected in any of the T-cell CLL cases, thus supporting the notion that T-cell CLL represents a malignancy of a mature phenotype. The one patient with lymphosarcoma whose tumor cells were TdT-positive subsequently developed T-cell acute lymphoblastic leukemia (ALL). Moreover, la-like antigen (p23,30) was detected on two of these tumor cell populations. In addition, it was shown that not all tumor cells were E-rosette-positive, since only cells from 3 of 4 patients were capable of forming spontaneous rosettes. These findings demonstrate that heteroantisera can provide an additional important tool for dissecting the heterogeneity of T-cell leukemias and for relating them to more differentiated normal T cells.

scholarly journals Circulating γδ T Cells in Response to Salmonellaenterica Serovar Enteritidis Exposure in Chickens

2006 ◽  
Vol 74 (7) ◽  
pp. 3967-3978 ◽  
Author(s):  
Angela Berndt ◽  
Jana Pieper ◽  
Ulrich Methner
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Type Strain ◽  
Wild Type Strain ◽  
Γδ T Cells ◽  
Immunohistochemical Analysis ◽  
Wild Type ◽  
Γδt Cells

ABSTRACT γδT cells are considered crucial to the outcome of various infectious diseases. The present study was undertaken to characterizeγδ (T-cell receptor 1+ [TCR1+]) T cells phenotypically and functionally in avian immune response. Day-old chicks were orally immunized with Salmonella enterica serovar Enteritidis live vaccine or S. enterica serovar Enteritidis wild-type strain and infected using the S. enterica serovar Enteritidis wild-type strain on day 44 of life. Between days 3 and 71, peripheral blood was examined flow cytometrically for the occurrence of γδ T-cell subpopulations differentiated by the expression of T-cell antigens. Three different TCR1+ cell populations were found to display considerable variation regarding CD8α antigen expression: (i) CD8α+high TCR1+ cells, (ii) CD8α+dim TCR1+ cells, and (iii) CD8α− TCR1+ cells. While most of the CD8α+high TCR1+ cells expressed the CD8αβ heterodimeric antigen, the majority of the CD8α+dim TCR1+ cells were found to express the CD8αα homodimeric form. After immunization, a significant increase of CD8αα+high γδ T cells was observed within the CD8α+high TCR1+ cell population. Quantitative reverse transcription-PCR revealed reduced interleukin-7 receptor α (IL-7Rα) and Bcl-x expression and elevated IL-2Rα mRNA expression of the CD8αα+highγδ T cells. Immunohistochemical analysis demonstrated a significant increase of CD8α+ and TCR1+ cells in the cecum and spleen and a decreased percentage of CD8β+ T cells in the spleen after Salmonella immunization. After infection of immunized animals, immune reactions were restricted to intestinal tissue. The study showed that Salmonella immunization of very young chicks is accompanied by an increase of CD8αα+high γδ T cells in peripheral blood, which are probably activated, and thus represent an important factor for the development of a protective immune response to Salmonella organisms in chickens.

DAP10 Costimulates Chimeric Receptor-Mediated T Cell Responses to Tumor Antigen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3052-3052
Author(s):  
Bianca Altvater ◽  
Sibylle Pscherer ◽  
Heribert Juergens ◽  
Claudia Rossig
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
T Cell Activation ◽  
Adoptive Immunotherapy ◽  
Peripheral Blood ◽  
Cell Activation ◽  
Human Peripheral Blood ◽  
Cytokine Secretion ◽  
Immunotherapy Of Cancer

Abstract Chimeric receptors (chRecs) combining extracellular recognition domains with the T cell receptor ζ an redirect the cellular immune response of primary T-cells to tumor cells. T cell activation by chRec induces efficient cytokine release and cytotoxicity, however, it fails to mediate proliferative responses, limiting the usefulness of chRec-gene-modified T cells for adoptive immunotherapy of cancer. Inclusion of a CD28 costimulatory signaling component in the chRec endodomain enhances antigen-specific proliferation. Whereas the signal mediated by ligation of CD28 is of crucial importance for the activation of resting CD4+ T cells, further molecules with costimulatory functions have contributory roles. NKG2D is a stimulatory receptor that was first identified in NK cells, but is also expressed in cytotoxic T cells and positively modulates CD8+ T cell immune responses. We hypothesized that inclusion of the NKG2D-associated signaling domain DAP10 would enhance the capacity of chRecs to induce tumor-specific activation and proliferation of in vitro expanded effector T cells. Based on a GD2-specific scFv, we generated chRecs containing either the DAP10 signaling chain alone (14.G2a-DAP10) or combined with TCRζ 14.G2a-DAP10ζ), and expressed them in nonspecifically activated human peripheral blood T cells of three individual donors by retroviral gene transfer. As controls, T cells were transduced with 14.G2a-ζ and -CD28ζ chRec. High chRec surface expression was obtained with all four constructs (55±11%, ζ; 85±3, CD28ζ; 68±5%, DAP10; 78±1%; DAP10ζ). Immunophenotypes were dominated by a CD3+CD8+ population in all cell cultures. Whereas DAP10 alone failed to mediate specific tumor cell lysis, 51Cr release assays revealed efficient and comparable lysis of GD2+ tumor targets by T cells transduced with all ζ-containing constructs, with 49±8% (ζ), 52±7% (CD28ζ), and 52±18% (DAP10ζ) cytolysis at an effector-to-target ratio of 40:1. Intracellular cytokine secretion by chRec+ T cells was induced in response to tumor targets by 14.G2a-ζ (up to 37% IFN-γ secreting cells), CD28ζ, and DAPζ (both up to 22%), but not by DAP10 alone (0,2%). Weekly stimulation with tumor cells for 6 weeks induced only limited expansion of T cells transduced with 14.G2a-ζ (7–45fold) or with 14.G2a-DAP10 (14–26-fold). Adding CD28 or DAP10 domains significantly enhanced expansion by a comparable degree (270–483-fold and 126–436-fold, respectively). Thus, while neither CD28 nor DAP10 enhances antigen-specific cytokine secretion and cytolysis, DAP10 signaling can completely replace CD28 signaling in costimulating antigen-specific proliferation of peripheral blood T cells. DAP10-containing chRec may be a powerful new tool for adoptive immunotherapy of cancer.

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