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Volume 17, Issue 9, Pages 1280-1291 (September 2015)
T cells specific for different latent and lytic viral proteins efficiently control Epstein-Barr virus–transformed B cells Justyna Nowakowska, Claudia Stuehler, Adrian Egli, Manuel Battegay, Georg Rauser, Glenn Robert Bantug, Christian Brander, Christoph Hess, Nina Khanna Cytotherapy Volume 17, Issue 9, Pages (September 2015) DOI: /j.jcyt Copyright © 2015 International Society for Cellular Therapy Terms and Conditions
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Figure 1 EBVmix is more immunogenic than commercially available peptide pools. EBV-specific memory T-cell frequency in healthy individuals (A), and HSCT recipients 30 to 108 months after HSCT (B). IFN-γ response of PBMCs was assessed by means of ELISpot after overnight stimulation with indicated peptide pools and calculated as spot-forming cells (SFC) per 106 PBMCs. Control represents unstimulated PBMCs. Only values >100 SFC/1 × 106 PBMCs (on subtraction of control) were defined as positive responses. Bars represent medians. Statistical significance (P) was determined with the use of KW with Dunn's multiple comparisons test (∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < ) (n ≥ 13 for healthy individuals and n = 9 for HSCT recipients). Cytotherapy , DOI: ( /j.jcyt ) Copyright © 2015 International Society for Cellular Therapy Terms and Conditions
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Figure 2 EBVmix is superior to single peptide pools in enrichment of EBV-specific CD4+ and CD8+ T cells. IFN-γ–based enrichment was performed after 4-h stimulation of PBMCs with peptide pools with the use of the CCS (n = 5). The fraction of CD4+IFN-γ+ (left) and CD8+IFN-γ+ (right) cells of total lymphocytes in the isolated IFN-γ–positive cells is shown. Control values of unstimulated PBMC (ranging from 0% to 1.35%) were subtracted. Bars represent medians. Statistical significance (P) was determined with the use of MWU (∗P < 0.05; ∗∗P < 0.01). Cytotherapy , DOI: ( /j.jcyt ) Copyright © 2015 International Society for Cellular Therapy Terms and Conditions
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Figure 3 Peptide-specific T-cell lines recognize EBV-infected cells and are not alloreactive. (A) Specificity of the T-cell lines after 2-week expansion in vitro. Intracellular IFN-γ production of the expanded EBV-specific T cells on 6-h re-stimulation with peptide pools (0.5 μg/peptide/mL) was analyzed by means of flow cytometry and are shown for CD4+ and CD8+ cells (left and right, respectively); background values of unstimulated controls (median, 0.2; range, 0.01–1.51) were subtracted. (B) IFN-γ response of T-cell lines to LCLs. T cells were incubated overnight with autologous LCLs (1:5 E:T ratio) and IFN-γ production measured by ELISpot; background values of unstimulated T cells (median, 33.3; range, 0–9200) and of control LCL (median, 1834; range, 50–9800) were subtracted; mean values from at least two independent experiments performed in duplicates are shown for each donor. All bars indicate medians. Values for all comparisons are P > 0.05 (MWU). (C) Alloreactivity of T-cell lines. Mixed lymphocyte reaction was performed with two donors and two different third-party DCs. [3H]-thymidine incorporation was measured after 96-h co-culture. Medians and ranges of at least two independent experiments performed in triplicates are shown. Differences between PBMCs and each cell line for both allo-DCs are statistically significant (P ≤ , MWU). Cytotherapy , DOI: ( /j.jcyt ) Copyright © 2015 International Society for Cellular Therapy Terms and Conditions
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Figure 4 EBVmix-specific T-cell lines are superior in controlling EBV-infected B cells over 4 weeks. (A) Short-term cytotoxicity of T-cell lines. T-cell lines were incubated for 6 h with autologous LCLs at 5:1 and 30:1 E:T ratio, and cytotoxicity was determined by means of flow cytometry with the use of Annexin V and PI staining. Where indicated, LCLs were pre-loaded for 1.5 h with a cognate antigen (0.5 μg/peptide/mL). Statistical significance (P) was determined with the use of MWU (∗P < 0.05; ∗∗P < 0.01). (B) Long-term capacity of EBV-specific T cells to control autologous LCLs. T cells were co-cultured with LCLs at E:T ratios from 1:2 to 32:1. After 4 weeks, the outgrowth of LCLs was assessed. The readout was defined as the lowest E:T ratio inhibiting the outgrowth of LCLs. The score equal to 0.25 and 64 indicates inhibition below and above the investigated range of E:T ratios (indicated by dotted lines), respectively; mean values from at least two independent experiments performed in triplicates are shown for each donor. Bars represent medians. Cytotherapy , DOI: ( /j.jcyt ) Copyright © 2015 International Society for Cellular Therapy Terms and Conditions
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Figure 5 The CD4-lines have high specificity to cognate antigens but show lower IFN-γ production and controlling capacity in response to LCLs than the T-cell lines. (A) Recognition of cognate peptide pools by CD4-lines. Intracellular production of IFN-γ after 6-h re-stimulation with indicated pools (0.5 μg/peptide/mL) was analyzed by means of flow cytometry; background values of unstimulated controls (median, 0.2; range, 0.05–3.44) were subtracted. Mean values from at least two independent experiments for each donor are shown. (B) Recognition of naturally processed antigens by CD4-lines. T cells were incubated overnight with autologous LCLs (E:T ratio 1:5) and IFN-γ response measured by ELISpot; background values of unstimulated T cells (median, 16.7; range, 0–2100) and of control LCLs (median, 1213; range, 25–4050) were subtracted. Mean values from at least two independent experiments performed in duplicates are shown for each donor. Bars represent medians. (C) Long-term capacity of CD4- and CD8-lines to control autologous LCLs. T cells were co-cultured with LCLs at E:T ratios from 1:2 to 32:1. After 4 weeks, the outgrowth of LCLs was assessed. The readout was defined as described in Figure 3B; mean values from at least two independent experiments performed in triplicates are shown for each donor. Bars represent medians. Cytotherapy , DOI: ( /j.jcyt ) Copyright © 2015 International Society for Cellular Therapy Terms and Conditions
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Figure 6 CD4-lines unable to control LCLs produce significantly less cytotoxic effector mediators because of their lower LCL recognition. Viability and PD-1 expression (A) and production of perforin, granzyme B and granulysin (B) of CD4-lines were assessed by means of flow cytometry at day 2 of co-culture with LCLs at 1:2 E:T ratio. Combined data from lines specific to EBVmix, LMP2a and EBNA1 of three donors are shown and stratified into LCL controllers (ctrl) (n = 2 for LMP2a and EBVmix) and LCL non-controllers (non-ctrl) (n = 1 for LMP2a and EBVmix, n = 3 for EBNA1); viability was determined with the use of Zombie Aqua Fixable Viability Kit; mean fluorescence intensity (MFI) values for respective isotype controls were subtracted. Bars represent medians. (C) IFN-γ response of ctrl or non-ctrl CD4-lines to stimulation with peptide or LCL (1:5 E:T ratio) were determined by means of IFN-γ ELISpot; background values of unstimulated T cells (median, 16.7; range, 0–2100) and of control LCL (median, 1213; range, 25–4050) were subtracted. Statistical significance (P) was determined with the use of MWU (∗P < 0.05; ∗∗∗P < 0.001). Cytotherapy , DOI: ( /j.jcyt ) Copyright © 2015 International Society for Cellular Therapy Terms and Conditions
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Supplementary Figure 1 EBV-specific T-cell lines expand in median 159-fold within 2 weeks. Expansion of EBV-specific IFN-γ–positive T-cell fractions is depicted. After 2-week expansion in vitro, the total number of cells in EBV-specific lines was determined and is shown along with the isolated cell numbers before expansion. Cytotherapy , DOI: ( /j.jcyt ) Copyright © 2015 International Society for Cellular Therapy Terms and Conditions
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Supplementary Figure 2 Expanded EBV-specific T-cell lines show mainly the effector memory phenotype. After 2-week expansion in vitro, the phenotype of T-cell lines was analyzed by means of flow cytometry to distinguish the central (CD45RO+CD62L+CCR+) and the effector (CD45RO+CD62L−CCR−) memory cells. Cytotherapy , DOI: ( /j.jcyt ) Copyright © 2015 International Society for Cellular Therapy Terms and Conditions
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Supplementary Figure 3 Peptide loading increases the recognition of LCL by EBV-specific T-cell lines. Proliferation of T cells after 96-h co-culture with autologous LCL or peptide-loaded autologous LCLs was measured by means of [3H]-thymidine incorporation; background values of T cells alone (median, 458; range, 0–4050) and LCL alone (median, 632; range, 535–803) were subtracted. Median with range values for two donors from at least two independent experiments performed in triplicates are shown. P values for all comparisons between the peptide pools >0.05 (MWU). Cytotherapy , DOI: ( /j.jcyt ) Copyright © 2015 International Society for Cellular Therapy Terms and Conditions
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