CD56+ human blood dendritic cells effectively promote TH1-type γδ T-cell responses

Blood ◽  
2009 ◽  
Vol 114 (20) ◽  
pp. 4422-4431 ◽  
Author(s):  
Georg Gruenbacher ◽  
Hubert Gander ◽  
Andrea Rahm ◽  
Walter Nussbaumer ◽  
Nikolaus Romani ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Human Blood ◽  
Γδ T Cells ◽  
Rapid Expansion ◽  
Γδ T Cell ◽  
Hla Dr ◽  
Tnf Α ◽  
Ifn Γ

Abstract CD56+ human dendritic cells (DCs) have recently been shown to differentiate from monocytes in response to GM-CSF and type 1 interferon in vitro. We show here that CD56+ cells freshly isolated from human peripheral blood contain a substantial subset of CD14+CD86+HLA-DR+ cells, which have the appearance of intermediate-sized lymphocytes but spontaneously differentiate into enlarged DC-like cells with substantially increased HLA-DR and CD86 expression or into fully mature CD83+ DCs in response to appropriate cytokines. Stimulation of CD56+ cells containing both DCs and abundant γδ T cells with zoledronate and interleukin-2 (IL-2) resulted in the rapid expansion of γδ T cells as well as in IFN-γ, TNF-α, and IL-1β but not in IL-4, IL-10, or IL-17 production. IFN-γ, TNF-α, and IL-1β production were almost completely abolished by depleting CD14+ cells from the CD56+ subset before stimulation. Likewise, depletion of CD14+ cells dramatically impaired γδ T-cell expansion. IFN-γ production could also be blocked by neutralizing the effects of endogenous IL-1β and TNF-α. Conversely, addition of recombinant IL-1β, TNF-α, or both further enhanced IFN-γ production and strongly up-regulated IL-6 production. Our data indicate that CD56+ DCs from human blood are capable of stimulating CD56+ γδ T cells, which may be harnessed for immunotherapy.

scholarly journals Activation of Human γδ T Cells: Modulation by Toll-Like Receptor 8 Ligands and Role of Monocytes

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 713 ◽  
Author(s):  
Ruben Serrano ◽  
Daniela Wesch ◽  
Dieter Kabelitz
Keyword(s):  
T Cells ◽  
T Cell ◽  
Zoledronic Acid ◽  
T Cell Activation ◽  
Cell Activation ◽  
Γδ T Cells ◽  
Γδ T Cell ◽  
Toll Like Receptor ◽  
Tnf Α ◽  
Ifn Γ

Background: Human Vγ9Vδ2 γδ T cells can kill a variety of cancer cells and have attracted substantial interest for cancer immunotherapy. Toll-like receptor (TLR) ligands are promising adjuvants for cancer immunotherapy, but TLR7/8 ligand Resiquimod has been shown to inhibit CD4 T-cell activation in a monocyte-dependent manner. Therefore, we studied the modulation of human γδ T-cell activation by TLR7/8 ligands. Methods: Peripheral blood mononuclear cells (PBMC) or purified γδ T cells together with purified monocytes were stimulated with zoledronic acid or phosphoantigens in the absence or presence of various imidazoquinoline TLR7 or TLR8 agonists. Read-out systems included interferon-γ induction and cellular expansion of γδ T cells, as well as viability, cell surface antigen modulation, and IL-1β and TNF-α production of monocytes. Results: TLR8 ligand TL8-506 and TLR7/8 ligand Resiquimod (but not TLR7 ligands) rapidly induced IFN-γ expression in γδ T cells within PBMC, and co-stimulated phosphoantigen-induced IFN-γ expression in γδ T cells. On the other hand, TLR8 ligands potently suppressed γδ T-cell expansion in response to zoledronic acid and phosphoantigen. Purified monocytes secreted large amounts of IL-1β and TNF-α when stimulated with TLR8 ligands but simultaneously underwent substantial cell death after 24 h. Conclusions: TLR8 ligand-activated monocytes potently co-stimulate early γδ T-cell activation but failed to provide accessory cell function for in vitro expansion of γδ T cells.

γδ T Cell-Mediated Immunotherapy of Malignancies with Zoledronate and IL-2.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2439-2439 ◽  
Author(s):  
Volker Kunzmann ◽  
Manfred Smetak ◽  
Brigitte Kimmel ◽  
Florian Weissinger ◽  
Karin Weigang-Koehler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Low Dose ◽  
Hematological Malignancies ◽  
Γδ T Cells ◽  
Γδ T Cell ◽  
Hla Dr ◽  
Ifn Γ

Abstract Despite major advances in our understanding of the adaptive immunity towards tumors and the introduction of vaccine-based strategies, durable responses are rare and adaptive immunotherapeutic approaches are still not an established treatment modality. Several lines of evidence indicate that MHC-independent effector cells of the innate immune system such as natural killer (NK) cells or γδ T cells significantly contribute to the immune surveillance of tumors. As we have shown previously, aminobisphosphonates (ABP) such as pamidronate or zoledronate are potent γδ T cell stimulatory compounds by inducing secretion of proinflammatory cytokines (i.e. IFN-γ) and cell-mediated cytotoxicity against lymphoma and myeloma cells in vitro. The detection of ABP as γδ T cell stimulating drugs at pharmacologically achievable concentrations in humans opened the possibility to evaluate the consequences of selective γδ T cell stimulation in vivo. The concept of γδ T cell-mediated immunotherapy is currently validated in a Phase II clinical trial with zoledronate (4mg i.v., d 1) and low dose IL-2 (2 x 106 IU/m2 s.c., d 1–6) for patients with hematological (NHL, myeloma, AML) and non-hematological malignancies (renal cell carcinoma and malignant melanoma). The results of our first clinical pilot study with pamidronate/IL-2 in patients with lymphoid malignancies showed that selective activation and expansion of γδ T cells can be induced in vivo. However, 50% of patients with hematological malignancies failed to respond to pamidronate/IL-2 in vitro. Therefore, positive in vitro sensitivity testing was an essential inclusion criterion in this trial. Immunomonitoring of the first 12 patients included in the study showed that zoledronate/IL-2 is highly effective in activating and expanding γδ T cells in vivo (104 TCRδ2+/HLA-DR+ cells/μl (range 11-323) at day 8 of cycle 1 compared to 3 TCRδ2+/HLA-DR+ cells (range 0-15) before treatment). In addition, IFN-γ serum levels increased from 7 to 110 pg/ml (mean 44, n=6) at day 2 at cycle 1 (day 0: 2, range 0–5). So far, objective tumor responses were observed only in hematological malignancies (updated data will be presented). The application of zoledronate/IL-2 is generally well tolerated. In conclusion, effective γδ T cell activation/expansion can be achieved in vivo by the combination of zoledronate and low dose IL-2. Because of the potent anti-tumor effects of γδ T cells this strategy might be a new attractive immunotherapy approach for malignancies with preserved γδ T cell function.

scholarly journals Differential Activation of V γ4V δ1 and V γ9V δ2 Cord Blood Derived Gamma Delta T Cells upon Exposure to EBV-Lcl and K562 Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2790-2790
Author(s):  
Jeremy Wee Kiat Ng ◽  
Joey Lai ◽  
Tony Kiat Hon Lim ◽  
William YK Hwang ◽  
Shang Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Single Cell ◽  
Γδ T Cells ◽  
Rapid Expansion ◽  
Leukemia Cell Line ◽  
Γδ T Cell ◽  
Gamma Delta

Abstract Gamma-delta (γδ) T cells have emerged as a promising candidate for adoptive cellular immunotherapy. To harness and maximize the anti-leukemia properties of these cells, we sort to comprehensively profile the transcriptomic signatures and immune repertoire of in vitro expanded γδ T cell products. Given the reported diverse TCR γδ repertoire and naïve nature of γδ T cells found in human cord blood (CB γδ), we serially track the molecular and cellular changes in these cells upon activation in expansion cultures. Based on the established viral reactivities of γδ T cell as well as prior studies showing their cross reactivities against leukemia and cancer cells, we had previously shown that stimulating CB γδ with an irradiated EBV-LCL feeder cell-based rapid expansion protocol (REP) is capable of generating cell products with potent and specific cytotoxicity against human AML cells. In the present study, using single cell RNA sequencing (scRNA-seq) coupled with single cell TCR γδ repertoire analysis, we compared the transcription signatures between our REP expanded γδ T cell (REP γδ) and non-manipulated γδ T cells reported in literatures, showing the progressive acquisition of an adult PB derived γδ T cell (PB γδ)-like cell states. Time course analysis demonstrated complex T cell activation and maturation trajectories correlating with variable level of clonal induction throughout the course of in vitro expansion. At the end of expansion, the harvested REP γδ are predominantly of the V γ4V δ1 subtype. Nevertheless, upon exposing REP γδ to target leukemia cell line K562, outgrowth of other non-V γ4V δ1 as well as the semi-invariant V γ9V δ2 cells were observed. Taken together, our data shows that as CB γδ expand and differentiate in culture, they adopt an adult PB γδ-like program. More importantly, our data highlights the rich clonal composition of in vitro expanded CB γδ, with different clonotypes being variably activated upon exposure to different stimuli. Such characteristics can potentially overcome the challenges of cancer heterogeneity and cell persistence, with the potential of improving outcomes in cell immunotherapy. Disclosures No relevant conflicts of interest to declare.

Quantitative and Functional Alterations of the Gamma Delta T-Cell Subset within Follicular Lymphoma Microenvironment.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2601-2601
Author(s):  
Sophie de Guibert ◽  
Jean-Baptiste Thibert ◽  
Céline Bonnaventure ◽  
Patricia Ame-Thomas ◽  
Céline Pangault ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Follicular Lymphoma ◽  
Peripheral Blood ◽  
Γδ T Cells ◽  
Γδ T Cell ◽  
Ifn Γ ◽  
Nonpeptide Antigens

Abstract T cells carrying a γδ TCR account for less than 5% of CD3pos T cells in healthy individuals but are key effectors of innate immunity through the recognition of some unprocessed nonpeptide antigens of both self and foreign origin. Whereas the Vδ2 subpopulation represents more than 70% of peripheral blood γδ T cells, the Vδ1 subset is mainly located in the mucosal tissue. Increasing evidence suggest that γδ T cells have potent antitumor activity and are implicated in the defense against some haematological and epithelial malignancies. Moreover, Vδ2 T cells constitute an attractive immunotherapy strategy since they could be expanded and activated both in vivo and in vitro using synthetic phosphoantigens and aminobiphosphonates. Such strategies are currently tested in preliminary clinical trials, notably in follicular lymphoma (FL). However, an exhaustive phenotypic and functional characterisation of γδ T cells in this disease, including tumor infiltration, is still lacking. We first explored the composition of FL microenvironment using a multicolour flow cytometry analysis. We observed a significant decrease in the percentage of myeloid (LinnegCD11cposHLADRpos) and plasmacytoid (LinnegCD123posHLADRpos) dendritic cells (P = .0011 and P < .0001, respectively) in FL compared to normal secondary lymphoid organs. In addition, among CD3pos T cells, the proportion of follicular helper T cells (CD4posCXCR5posICOShi) was increased (P = .001) whereas regulatory T-cell (CD4posCD25posfoxp3pos) frequency was not altered. When considering the γδ T-cell compartment, we first highlighted a reduction of the Vδ2 subset in normal tonsils (Vδ2 = 23.48 ± 0.15% of γδ T cells, n = 11) when compared with peripheral blood. Remaining non-δ2 γδT cells were predominantly δ1 T cells. More importantly, infiltrating γδ T cells were significantly decreased in lymph node biopsies from FL patients (mean = 0.48 ± 0.4% of CD3pos T cells; n = 27) when compared both to normal tonsils (mean = 2.49 ± 1.6% of CD3pos T cells; n = 33) (P < .0001) and reactive lymph nodes (mean = 2.64 ± 2.6% of CD3pos T cells; n = 9) (P = .0009). This reduction affected both the Vδ1 and Vδ2 T-cell subsets. The functionality of γδ T cells was then assessed by the measurement of cell expansion and production of IFN-γ upon stimulation with the isopentenyl pyrophosphate (IPP) phosphoantigen. Amplification rate in vitro reached 14.6 ± 4.6 fold in tonsils (n = 10) but only 4.36 ± 1.9 fold in FL samples (n = 7) (P < .002) after 5 days of culture in the presence of IPP + IL-2 + IL-15. When focusing on the δ2 subset, this difference was further increased with a 40-fold amplification in tonsil and a 3-fold amplification in FL samples (P = .0004). Evaluation of IFN-γ production using ELISPOT assay revealed a high heterogeneity among tumor samples since 1 to 40% of δ2 T cells were able to respond to IPP stimulation (n = 7). Preliminary data argued for an association between the quantity and the functionality of γδ T cells in FL tumors. In conclusion, we reported an alteration of γδ T cell frequency and functionality within FL tumor niche. The next purpose will be to correlate these in vitro defects with in vivo clinical responses to immunotherapy strategies targeting γδ T cells.

scholarly journals Genetic models reveal origin, persistence and non-redundant functions of IL-17–producing γδ T cells

2018 ◽  
Vol 215 (12) ◽  
pp. 3006-3018 ◽  
Author(s):  
Inga Sandrock ◽  
Annika Reinhardt ◽  
Sarina Ravens ◽  
Christoph Binz ◽  
Anneke Wilharm ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Diphtheria Toxin ◽  
Γδ T Cells ◽  
Γδ T Cell ◽  
Fetal Thymus ◽  
Effector Cells ◽  
Cell Functions ◽  
Ifn Γ ◽  
Redundant Functions

γδ T cells are highly conserved in jawed vertebrates, suggesting an essential role in the immune system. However, γδ T cell–deficient Tcrd−/− mice display surprisingly mild phenotypes. We hypothesized that the lack of γδ T cells in constitutive Tcrd−/− mice is functionally compensated by other lymphocytes taking over genuine γδ T cell functions. To test this, we generated a knock-in model for diphtheria toxin–mediated conditional γδ T cell depletion. In contrast to IFN-γ–producing γδ T cells, IL-17–producing γδ T cells (Tγδ17 cells) recovered inefficiently after depletion, and their niches were filled by expanding Th17 cells and ILC3s. Complementary genetic fate mapping further demonstrated that Tγδ17 cells are long-lived and persisting lymphocytes. Investigating the function of γδ T cells, conditional depletion but not constitutive deficiency protected from imiquimod-induced psoriasis. Together, we clarify that fetal thymus-derived Tγδ17 cells are nonredundant local effector cells in IL-17–driven skin pathology.

scholarly journals Single-cell analysis reveals the origins and intrahepatic development of liver-resident IFN-γ-producing γδ T cells

2021 ◽  
Vol 18 (4) ◽  
pp. 954-968
Author(s):  
Yuan Hu ◽  
Keke Fang ◽  
Yanan Wang ◽  
Nan Lu ◽  
Haoyu Sun ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Adoptive Transfer ◽  
Γδ T Cells ◽  
Developmental Trajectory ◽  
Γδ T Cell ◽  
Developmental Pathway ◽  
Hematopoietic Progenitor ◽  
Ifn Γ

Abstractγδ T cells are heterogeneous lymphocytes located in various tissues. However, a systematic and comprehensive understanding of the origins of γδ T cell heterogeneity and the extrathymic developmental pathway associated with liver γδ T cells remain largely unsolved. In this study, we performed single-cell RNA sequencing (scRNA-seq) to comprehensively catalog the heterogeneity of γδ T cells derived from murine liver and thymus samples. We revealed the developmental trajectory of γδ T cells and found that the liver contains γδ T cell precursors (pre-γδ T cells). The developmental potential of hepatic γδ T precursor cells was confirmed through in vitro coculture experiments and in vivo adoptive transfer experiments. The adoptive transfer of hematopoietic progenitor Lin−Sca-1+Mac-1+ (LSM) cells from fetal or adult liver samples to sublethally irradiated recipients resulted in the differentiation of liver LSM cells into pre-γδ T cells and interferon-gamma+ (IFN-γ+) but not interleukin-17a+ (IL-17a+) γδ T cells in the liver. Importantly, thymectomized mouse models showed that IFN-γ-producing γδ T cells could originate from liver LSM cells in a thymus-independent manner. These results suggested that liver hematopoietic progenitor LSM cells were able to differentiate into pre-γδ T cells and functionally mature γδ T cells, which implied that these cells are involved in a distinct developmental pathway independent of thymus-derived γδ T cells.

scholarly journals Critical role for Slam/SAP signaling in the thymic developmental programming of IL-17- and IFN-γ-producing γδ T cells

2019 ◽  
Author(s):  
Oliver Dienz ◽  
Victoria L. DeVault ◽  
Shawn C. Musial ◽  
Somen K. Mistri ◽  
Linda Mei ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Signaling Pathway ◽  
Functional Programming ◽  
Γδ T Cells ◽  
T Cell Subsets ◽  
Γδ T Cell ◽  
Thymic Development ◽  
Slam Family ◽  
Ifn Γ

AbstractDuring thymic development, γδ T cells commit to either an IFN-γ- or an IL-17-producing phenotype through mechanisms that remain unclear. Here, we investigated whether the SLAM/SAP signaling pathway played a role in the functional programming of thymic γδ T cells. Characterization of SLAM family receptor expression revealed that thymic γδ T cell subsets were each marked by distinct co-expression profiles of SLAMF1, SLAMF4, and SLAMF6. In the thymus, immature CD24hiVγ1 and Vγ4 γδ T cells were largely contained within a SLAMF1+SLAMF6+double positive (DP) population, while mature CD24lowsubsets were either SLAMF1+or SLAMF6+single positive (SP) cells. In the periphery, SLAMF1 and SLAMF6 expression on Vγ1, Vγ4, and Vγ6 T cells distinguished IL-17- and IFN-γ-producing subsets, respectively. Disruption of SLAM family receptor signaling through deletion of SAP resulted in impaired thymic γδ T cell maturation at the CD24hiSLAMF1+SLAMF6+DP stage that was associated with a decreased frequency of CD44+RORγt+γδ T cells. These defects were in turn associated with impaired γδ T cell IL-17 and IFN-γ production in both the thymus as well as in peripheral tissues. The role for SAP was subset-specific, as Vγ1, Vγ4, Vγ5, but not Vγ6 subsets were SAP-dependent. Together, these data suggest that the SLAM/SAP signaling pathway regulates a critical checkpoint in the functional programming of IL-17 and IFN-γ-producing γδ T cell subsets during thymic development.

scholarly journals γδ T Cells but Not NK Cells Are Essential for Cell-Mediated Immunity against Plasmodium chabaudi Malaria

2010 ◽  
Vol 78 (10) ◽  
pp. 4331-4340 ◽  
Author(s):  
William P. Weidanz ◽  
GayeLyn LaFleur ◽  
Andrew Brown ◽  
James M. Burns ◽  
Irene Gramaglia ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Γδ T Cells ◽  
Cell Mediated Immunity ◽  
Γδ T Cell ◽  
Plasmodium Chabaudi ◽  
Ifn Γ ◽  
Time Courses

ABSTRACT Blood-stage Plasmodium chabaudi infections are suppressed by antibody-mediated immunity and/or cell-mediated immunity (CMI). To determine the contributions of NK cells and γδ T cells to protective immunity, C57BL/6 (wild-type [WT]) mice and B-cell-deficient (JH−/− ) mice were infected with P. chabaudi and depleted of NK cells or γδ T cells with monoclonal antibody. The time courses of parasitemia in NK-cell-depleted WT mice and JH−/− mice were similar to those of control mice, indicating that deficiencies in NK cells, NKT cells, or CD8+ T cells had little effect on parasitemia. In contrast, high levels of noncuring parasitemia occurred in JH−/− mice depleted of γδ T cells. Depletion of γδ T cells during chronic parasitemia in B-cell-deficient JH−/− mice resulted in an immediate and marked exacerbation of parasitemia, suggesting that γδ T cells have a direct killing effect in vivo on blood-stage parasites. Cytokine analyses revealed that levels of interleukin-10, gamma interferon (IFN-γ), and macrophage chemoattractant protein 1 (MCP-1) in the sera of γδ T-cell-depleted mice were significantly (P < 0.05) decreased compared to hamster immunoglobulin-injected controls, but these cytokine levels were similar in NK-cell-depleted mice and their controls. The time courses of parasitemia in CCR2−/− and JH−/− × CCR2−/− mice and in their controls were nearly identical, indicating that MCP-1 is not required for the control of parasitemia. Collectively, these data indicate that the suppression of acute P. chabaudi infection by CMI is γδ T cell dependent, is independent of NK cells, and may be attributed to the deficient IFN-γ response seen early in γδ T-cell-depleted mice.

scholarly journals Human cytomegalovirus elicits fetal γδ T cell responses in utero

2010 ◽  
Vol 207 (4) ◽  
pp. 807-821 ◽  
Author(s):  
David Vermijlen ◽  
Margreet Brouwer ◽  
Catherine Donner ◽  
Corinne Liesnard ◽  
Marie Tackoen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Human Cytomegalovirus ◽  
Early Life ◽  
Γδ T Cells ◽  
In Utero ◽  
Γδ T Cell ◽  
Cmv Infection ◽  
Cell Responses ◽  
Ifn Γ

The fetus and infant are highly susceptible to viral infections. Several viruses, including human cytomegalovirus (CMV), cause more severe disease in early life compared with later life. It is generally accepted that this is a result of the immaturity of the immune system. γδ T cells are unconventional T cells that can react rapidly upon activation and show major histocompatibility complex–unrestricted activity. We show that upon CMV infection in utero, fetal γδ T cells expand and become differentiated. The expansion was restricted to Vγ9-negative γδ T cells, irrespective of their Vδ chain expression. Differentiated γδ T cells expressed high levels of IFN-γ, transcription factors T-bet and eomes, natural killer receptors, and cytotoxic mediators. CMV infection induced a striking enrichment of a public Vγ8Vδ1-TCR, containing the germline-encoded complementary-determining-region-3 (CDR3) δ1–CALGELGDDKLIF/CDR3γ8–CATWDTTGWFKIF. Public Vγ8Vδ1-TCR–expressing cell clones produced IFN-γ upon coincubation with CMV-infected target cells in a TCR/CD3-dependent manner and showed antiviral activity. Differentiated γδ T cells and public Vγ8Vδ1-TCR were detected as early as after 21 wk of gestation. Our results indicate that functional fetal γδ T cell responses can be generated during development in utero and suggest that this T cell subset could participate in antiviral defense in early life.

scholarly journals Responses of Bovine WC1+ γδ T Cells to Protein and Nonprotein Antigens of Mycobacterium bovis

2002 ◽  
Vol 70 (11) ◽  
pp. 6114-6120 ◽  
Author(s):  
Michael D. Welsh ◽  
Hilary E. Kennedy ◽  
Allister J. Smyth ◽  
R. Martyn Girvin ◽  
Peter Andersen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Mycobacterium Bovis ◽  
Γδ T Cells ◽  
T Cell Proliferation ◽  
Γδ T Cell ◽  
Ifn Γ ◽  
Antigenic Targets ◽  
Protein Versus

ABSTRACT WC1+ γδ T cells of Mycobacterium bovis-infected cattle are highly responsive to M. bovis sonic extract (MBSE). In mycobacterial infections of other species, γδ T cells have been shown to respond to protein and nonprotein antigens, but the bovine WC1+ γδ T-cell antigenic targets within MBSE require further definition in terms of the dominance of protein versus nonprotein components. The present study sought to characterize the WC1+ γδ T-cell antigenic targets, together with the role of interleukin-2 (IL-2), in the context of M. bovis infection. This was achieved by testing crude and defined antigens to assess protein versus nonprotein recognition by WC1+ γδ T cells in comparison with CD4+ αβ T cells. Both cell types proliferated strongly in response to MBSE, with CD4+ T cells being the major producers of gamma interferon (IFN-γ). However, enzymatic digestion of the protein in MBSE removed its ability to stimulate CD4+ T-cell responses, whereas some WC1+ γδ T-cell proliferation remained. The most antigenic protein inducing proliferation and IFN-γ secretion in WC1+ γδ T-cell cultures was found to be ESAT-6, which is a potential novel diagnostic reagent and vaccine candidate. In addition, WC1+ γδ T-cell proliferation was observed in response to stimulation with prenyl pyrophosphate antigens (isopentenyl pyrophosphate and monomethyl phosphate). High levels of cellular activation (CD25 expression) resulted from MBSE stimulation of WC1+ γδ T cells from infected animals. A similar degree of activation was induced by IL-2 alone, but for WC1+ γδ T-cell division IL-2 was found to act only as a costimulatory signal, enhancing antigen-driven responses. Overall, the data indicate that protein antigens are important stimulators of WC1+ γδ T-cell proliferation and IFN-γ secretion in M. bovis infection, with nonprotein antigens inducing significant proliferation. These findings have important implications for diagnostic and vaccine development.

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