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. 2009 Feb 17;106(7):2307-12.
doi: 10.1073/pnas.0810059106. Epub 2009 Jan 26.

Cross-presenting Human Gammadelta T Cells Induce Robust CD8+ Alphabeta T Cell Responses

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Free PMC article

Cross-presenting Human Gammadelta T Cells Induce Robust CD8+ Alphabeta T Cell Responses

Marlène Brandes et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Gammadelta T cells are implicated in host defense against microbes and tumors but their mode of function remains largely unresolved. Here, we have investigated the ability of activated human Vgamma9Vdelta2(+) T cells (termed gammadelta T-APCs) to cross-present microbial and tumor antigens to CD8(+) alphabeta T cells. Although this process is thought to be mediated best by DCs, adoptive transfer of ex vivo antigen-loaded, human DCs during immunotherapy of cancer patients has shown limited success. We report that gammadelta T-APCs take up and process soluble proteins and induce proliferation, target cell killing and cytokine production responses in antigen-experienced and naïve CD8(+) alphabeta T cells. Induction of APC functions in Vgamma9Vdelta2(+) T cells was accompanied by the up-regulation of costimulatory and MHC class I molecules. In contrast, the functional predominance of the immunoproteasome was a characteristic of gammadelta T cells irrespective of their state of activation. Gammadelta T-APCs were more efficient in antigen cross-presentation than monocyte-derived DCs, which is in contrast to the strong induction of CD4(+) alphabeta T cell responses by both types of APCs. Our study reveals unexpected properties of human gammadelta T-APCs in the induction of CD8(+) alphabeta T effector cells, and justifies their further exploration in immunotherapy research.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
γδ T-APCs cross-present soluble protein antigen to CD8+ αβ T cells. (A) γδ T-APCs and DCs were treated with PPD, then washed and cocultured for 10 days with CFSE-labeled bulk αβ T cells or purified naïve CD8+ αβ T cells at a APC/responder cell ratio of 1:10. Results in row 3 illustrate that the majority of proliferating CD45RO+ cells were CD8+ responder cells. Data are representative of 2 and 3 experiments with bulk and naive CD8+ αβ T cells, respectively. (B) γδ T-APCs (squares) and DCs (circles) cross-present influenza matrix protein M1 to the HLA-A2-restricted, M1p58–66-specific CD8+ αβ T cell clone FLUMA55 (APC/responder cell ratios varied between 1: 5 and 3:1 but this variation had no obvious effect on the results with FLUMA55). Negative control, 4 μM M1 treated, HLA-A2-negative B cells (triangles). The Right compiles data from 7 independent FLUMA55 cross-priming experiments with γδ T-APCs and DCs treated with 0.4 μM M1; additional control, 0.1 μM M1p58–66 pulsed DCs. Boxes' lower/upper ends and middle lines depict 25/75 percentile and median. (C) Bulk CD8+ αβ T cells were stimulated with M1 (filled squares) or M1p58–66 (open squares) treated γδ T-APCs and S/LPS-DCs (APC/responder cell ratio of 1:20), and, after 10 days of culture, M1p58–66-specific responder cells were quantified by M1p58–66-tetramer staining. (D) γδ T-APCs and DCs, either treated with shear force and LPS or with CD40L, differ in their efficiency to cross-present M1 to bulk CD8+ αβ T cells. Blood cells from 2 to 4 different donors; 1-tailed students t test; NS, not significant.
Fig. 2.
Fig. 2.
Cellular distribution of MHC I during activation of Vγ9Vδ2+ T cells. (A) Activation of Vγ9Vδ2+ T cells with IPP for 6–48 h in the presence of feeder B cells followed by confocal immunofluorescence microscopic analysis of Vδ2-TCR staining (green) in combination with digital interference contrast images (Upper), or with MHC I staining (fire scale color mapping) (Lower); 0 h, resting γδ T cells. Control, digital interference contrast images in combination with MHC I (red) and nuclei (blue) stainings in immature (iDC) and mature DCs (mDC). Bar graph represents quantifications of intracellular (cytosol) and cell membrane (surface) associated MHC I within individual γδ T cells at the indicated IPP stimulation time points; relative unit (RU) of 1 equals 106 counts with 3–6 cells analyzed per data point. (B) Cell surface expression of MHC I and MHC II was analyzed by flow cytometry in freshly isolated (nonstimulated) and activated Vδ2+ γδ T cells that were stimulated for 12 or 36 h with IPP. (C) Increased cell surface MHC I staining in γδ T cells involves de novo MHC I synthesis. MHC I (red) in conjunction with GM130 (green) is shown as maximum intensity projections in combination with digital interference contrast images (50:50 fluorescence intensity ratio in yellow). [Scale bars: 5 μm (10 μm for DCs).]
Fig. 3.
Fig. 3.
γδ T-APCs and DCs fail to cross-present Melan-A to Melp26–35-specific CD8+ αβ T cells. (A) γδ T-APCs were treated with or without Melan-A and then cocultured with the HLA-A2-restricted, Melp26–35-specific responder cell clone LAU 337 for determination of intracellular IFN-γ. Controls include Melp26–35-pulsed γδ T-APCs together with LAU 337 responder cells, and M1 cross-presenting γδ T-APCs together with FLUMA55 responder cells. Numbers in brackets represent the mean. (B) γδ T-APCs and DCs were incubated with Melan-A at indicated concentrations and cocultured with CFSE-labeled, HLA-A2-restricted blood CD8+ αβ T cells at a APC/responder cell ratio of 1:10. Alternatively, Melp26–35 pulsed γδ T-APCs or M1 cross-presenting γδ T-APCs were used and the numbers (percentage of total) of Melp26–35- and M1p58–66-tetramer positive responder cells were determined at 10 days of culture. Data are representative of 2–4 experiments.
Fig. 4.
Fig. 4.
Vγ9Vδ2+ T cells express highly active immunoproteasome. (A) Proteins in lysates of freshly isolated (resting) Vγ9Vδ2+ T cells or γδ T-APCs or monocyte-derived DCs (iDCs) or B cells (EBV-B) were separated by SDS/PAGE and analyzed by Western blot. α5, protease subunit present in both standard and immunoproteasome; β1i (LMP2), immunoproteasome-specific subunit; Αctin, protein loading control. (B) Purified proteasome from γδ T-APCs, monocyte-derived immature DCs (iDCs) and human embryonic epithelial cells (HEK293) were incubated at 37 °C for 16 h with the peptide substrate Melan-A15–40 and the peptide products were fractionated by reverse-phase HPLC and then identified by mass spectroscopy. The peaks at 20.3 min elution time contained the standard proteasome-specific peptide Melan-A15–35 (highlighted with gray bars). The yield in Melan-A15–35 was highest with proteasome preparations from HEK293 cells. Of note, the Melan-A15–35 was not detected with proteasome preparations from γδ T-APCs, suggesting dominant proteolytic activity by the immunoproteasome. Data are representative of 2 and 3 separate experiments.
Fig. 5.
Fig. 5.
Cross-presenting γδ T-APCs induce robust primary CD8+ αβ T cell responses. (A) γδ T cells and DCs, treated with 4 μM M1 (see Fig. 1C), were cultured with sorted naïve CD8+ αβ T cells (APC/responder cell ratio of 1:20) for 10 days (cycle 1), or were restimulated with M1 cross-presenting APCs and cultured for another 10 days (cycle 2). Responder cells were identified by M1p58–66-tetramer staining. (B) M1 cross-presenting γδ T-APCs and DCs (Left and Right, respectively) were used as APCs, and naïve CD8+ αβ T cell-derived responder cells were cloned by limited dilution culture. 51Cr-labeled target cells were pulsed with M1p58–66 (filled circles and squares) or unrelated Melp26–35 (open circles and squares) at indicated concentrations or were unpulsed and mixed at a 1:1 ratio with responder clones. One representative high-affinity (circles) and low-affinity (squares) responder clone are shown for each cytotoxic T cell cloning experiment; data are representative of 26 M1p58–66-tetramer+ T cell clones.

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