CD4 + CD25 high Foxp3 + T regulatory cells kill autologous CD8(+) and CD4(+) T cells using Fas/FasL- and Granzyme B- mediated pathways Laura Strauss, Christoph Bergmann, Theresa L. Whiteside University of Pittsburgh Cancer Institute and Departments of Pathology, Immunology and Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 FoxP3 +
Objectives of our study were: To define mechanisms employed by Treg to mediate suppression of proliferating T responder cells (RC) We considered 3 possible mechanisms: 1. Cytokine- mediated death. 2. Death receptor-mediated apoptosis. 3. Cytolysis mediated by granzymes/perforin.
Methods for studies of Treg Isolate CD4+CD25 high and CD4+CD25 neg or CD8+CD25 neg T cells from PBMC of NC or patients with cancer bv single-cell sorting (FACS) Multicolor flow cytometry: phenotype Suppressor function: CFSE-labeled RC (+ OKT3+ IL-2) + 5-day co-culture Unlabeled S Suppression of RC proliferation Laser + 7-AAD FLOCA (Flow cytometry-based cytotoxicity assay)
We have shown before that CD8 + T effector cells in the circulation of patients with HNSCC (but not NC) are highly sensitive to apoptosis 4,5 Apoptosis in fresh T cell subsets from HNSCC patient and NC fresh PBMC NC Annex V gated on CD3 + CD8 + CD25 - gated on CD3 + CD4 + CD25 -.3%.2% CD8 CD4 fresh PBMC HNSCC 72% 5.1% CD8 CD4 4 Reichert et al., 2 5 Hoffmann et al., 22
Treg mediate suppression of autologous CD4 + or CD8 + RC proliferation via direct cell-cell contact 6 7 % Treg in PBMC Gated on CD4 + CD25 high T cells CD4 + CD25 high + CD4 + CD25 - or CD8 + CD25 - co-cultures 9 p.3 6 8 % CD25 high cells 5 4 3 2 P.1 % Suppression 7 6 5 4 3 2 S:RC ratio:1:2 1 1 NC (n=15) HNSCC (n=35) non-tr n = 5 TR 6 Strauss et al., 27, Clinical Cancer Res, 1:13 (15):4352
Human CD4 + CD25 high Treg suppress proliferation and induce apoptosis in autologous CD8 + responder cells 5-day co-cultures in the presence of 15 IU/mL IL-2 + OKT3 Responder cells alone (RC) (CD8 + CD25 neg ) CD8 + (RC) cells (RC) cells + CD4 + CD25 high (S) S/RC=1:1 Events 3 6 9 12 89% HNSCC 6 7-AAD.3% 99.7% CD8 + (RC) cells 56% 24% 3 6 9 CFSE 12 CD8 + 2
Human CD4 + CD25 high Treg suppress proliferation but do not mediate apoptosis in autologous CD4 + responder cells 5-day co-cultures in the presence of 15 IU/mL IL-2 + OKT3 Responder cells alone (RC) (CD4 + CD25 neg ) CD4 + (RC) cells (RC) cells + CD4 + CD25 high (S) S/RC=1:1 Events 3 6 9 12 96% HNSCC 7-AAD 1.2% 98.8% CD4 + (RC) cells.2% 7-AAD Treg Tr cells 82% 77.4% 18.8% 15% 3 6 9 12 CFSE 99.8% CD4 + 8% 3% CD4 +
Expression of Fas and FasL on CD4 + CD25 T cells in NC or HNSCC patients CD25 high % positive cells 1 9 8 7 6 5 4 3 2 1 FRESH PBMC p.1 Fas NC (n=15) HNSCC (n=35) p.3 FasL Fas FasL Normal HNSCC Control gated on CD4 + CD25 high T cells 43% 95% 5.5% 92% FasL CD4
Treg can kill autologous CD8 + CD25 - RC but not CD4 + CD25 - RC via the Fas/FasL pathway + CFSE-labeled CD8 + RC CD4 + CD25 high Treg (FasL+) + CFSE-labeled CD4 + RC 56% 92% 2 92% 24% Events 68% Events 2 12% 7-AAD + anti-fasl- Ab 3 6 9 12 7-AAD + anti-fasl- Ab 3 6 9 12 % 1%.3% Events 62% 18% 72% Events 97% 99% 99.7 % CD8 3 6 9 12 CFSE CD4 1 3 6 9 12 CFSE 15 IU/mL IL-2 1 IU/mL IL-2 Results of cell death obtained in FLOCA
Expression of Granzymes and Perforin in fresh and activated peripheral T-cell T subsets A. MFI±SD Gated on CD4 + CD25 high in fresh PBMC 16 NC n=15 HNSCC n=25 14 * *p.1 12 1 8 6 * +OKT3, IL-2 (15 IU/mL) MFI±SD 16 14 12 1 8 6 Activated CD4 + CD25 high * NC n=15 HNSCC n=25 *p.3 * 4 4 2 2 Granzyme B Granzyme A Perforin Granzyme B Granzyme A Perforin B. Gated on CD4 + CD25 - in fresh PBMC 16 NC n=15 14 HNSCC n=25 16 14 Activated CD4 + CD25 high NC n=15 HNSCC n=25 12 12 MFI±SD 1 8 MFI±SD 1 8 6 6 4 4 2 2 Granzyme B Granzyme A Perforin Granzyme B Granzyme A Perforin
Expression of cytotoxins is regulated by IL-2 2 in the presence of the partner T cell (15 IU IL-2/mL) (1 IU IL-2/mL) CD4 + CD25 neg after co-incubation with Treg cells 1S:1 (RC) Granzyme B-PE 98% 97% 88% Granzyme A-PE Perforin-FITC Granzyme B-PE 25% 1% 15% Granzyme A-PE Perforin-FITC R CD4 + CD25 high after co-incubation with CD4+ RC cells 1S:1 (RC) Granzyme B-PE 3% 4% 7% Granzyme A-PE Perforin-FITC Granzyme B-PE CD4 99% PerCP Granzyme A-PE 92% 75% Perforin-FITC S 1 2 3 CD4 5-day co-cultures of Treg and autologous RC
Treg suppress CD4+ RC at low and high dose of IL-2, but can kill RC only at high IL-2 2 concentrations S/RC=1:1 (15 IU IL-2/mL) 96% HNSCC CD4 + (RC) cells Treg Tr cells 77.4% 82% 3 6 9 12.2% 99.8% 15% 18.8% 8% 3% 7-AAD Events 98% HNSCC CD4 + (RC) cells Treg (1 IU IL-2/mL) 78% 3 6 9 12 22% 1 CFSE CD4 At low IL-2 doses, Treg induce suppression of RC proliferation and then undergo apoptosis. This type of suppression does not involve death of RC.
Conclusion 1 Mechanisms responsible for RC death and Treg survival in these co-cultures are IL-2 dependent
Treg- mediated killing of CD4 + RC is GranzymeB- dependent, but Perforin-independent independent CD4 + (RC) cells Treg 1 IU/mL IL-2 98%.5% Data.22 64%.4% 3 6 9 12 35.5% 99.6% Events 7-AAD Granzyme B-PE 97% + sirna GrB 6% CD4 + (RC) cells Treg.2%.3% 3 6 9 12 CFSE 99.8% CD4 CD-4 Granzyme B-PE 12% 99.7%
CD4 + RC- mediated killing of Treg is GranzymeB and Perforin-dependent CD4 + (RC) cells Treg 15 IU/mL IL-2 82% Data.22.5%.4% 64% 3 6 9 12 99.6% 35.5% Events 7-AAD Perforin-FITC 97% 98% CD4 + (RC) cells +ConcanamycinA Treg 3 6 9 12 1 7% 93% CFSE Perforin-FITC 7% CD-4
Conclusion 2 The GranzymeB-mediated reciprocal killing mediated by RC or Treg is IL-2-dependent
Expression of proteins that protect from apoptosis is regulated by IL-2 Bcl-2 (MFI±SD) 2 18 16 14 12 1 8 6 4 2 Fresh PBMC * NC n=15 HNSCC n=35 * p.1 PI-9 expression in Treg and RC after co-culture + IL-2 PI-9 NC AD NED 1 IU/mL IL-2: MFI ± SD CD4 + CD25 high 83 ± 5* 115 ± 1* 15* ± 12 CD4 + CD25-25 ± 12 25 ± 1 32 ± 2.5 CD8 + CD25-12 ± 1.8 28 ± 4.5 65 ± 5.8 *p.1 for differences between Treg and RC 15 IU/mL IL-2: CD4 + CD25 high 22 ± 6.7* 18 ± 4.58* 18 ± 2.5* CD4 + CD25-85 ± 6.5 68 ± 12 58 ± 1 Foxp3 high Foxp3 interm. Foxp3 low CD8 + CD25-95 ± 5.9 64 ± 8 54 ± 12 *p.3 for differences between Treg and RC At low IL-2 concentrations, Treg co-cultured with RC do not up-regulate PI-9 and are sensitive to GrB-mediated apoptosis.
Conclusions Treg can suppress RC via three different mechanisms Tr1 largely use IL-1 and TGFβ1 to suppress RC proliferation (a contact independent process) CD8+RC expansion is suppressed by the Fas/FasLmediated apoptosis CD4+RC proliferation is suppressed via a contactdependent GrB/perforin pathway which is regulated by the IL-2 concentration in the microenvironment Resistance or sensitivity to death of Treg vs. RC is dependent on IL-2-mediated T cell activation