CD4 + CD25 high Foxp3 + T regulatory cells kill autologous CD8(+) and CD4(+) T cells using Fas/FasL- and Granzyme B- mediated pathways

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1 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 FoxP3 +

2 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.

3 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)

4 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

5 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 % CD25 high cells P.1 % Suppression 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

6 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 % HNSCC 6 7-AAD.3% 99.7% CD8 + (RC) cells 56% 24% CFSE 12 CD8 + 2

7 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 % HNSCC 7-AAD 1.2% 98.8% CD4 + (RC) cells.2% 7-AAD Treg Tr cells 82% 77.4% 18.8% 15% CFSE 99.8% CD4 + 8% 3% CD4 +

8 Expression of Fas and FasL on CD4 + CD25 T cells in NC or HNSCC patients CD25 high % positive cells 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

9 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 AAD + anti-fasl- Ab % 1%.3% Events 62% 18% 72% Events 97% 99% 99.7 % CD CFSE CD CFSE 15 IU/mL IL-2 1 IU/mL IL-2 Results of cell death obtained in FLOCA

10 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 * +OKT3, IL-2 (15 IU/mL) MFI±SD Activated CD4 + CD25 high * NC n=15 HNSCC n=25 *p.3 * 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= Activated CD4 + CD25 high NC n=15 HNSCC n= MFI±SD 1 8 MFI±SD Granzyme B Granzyme A Perforin Granzyme B Granzyme A Perforin

11 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 CD4 5-day co-cultures of Treg and autologous RC

12 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% % 99.8% 15% 18.8% 8% 3% 7-AAD Events 98% HNSCC CD4 + (RC) cells Treg (1 IU IL-2/mL) 78% % 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.

13 Conclusion 1 Mechanisms responsible for RC death and Treg survival in these co-cultures are IL-2 dependent

14 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% % 99.6% Events 7-AAD Granzyme B-PE 97% + sirna GrB 6% CD4 + (RC) cells Treg.2%.3% CFSE 99.8% CD4 CD-4 Granzyme B-PE 12% 99.7%

15 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% % 35.5% Events 7-AAD Perforin-FITC 97% 98% CD4 + (RC) cells +ConcanamycinA Treg % 93% CFSE Perforin-FITC 7% CD-4

16 Conclusion 2 The GranzymeB-mediated reciprocal killing mediated by RC or Treg is IL-2-dependent

17 Expression of proteins that protect from apoptosis is regulated by IL-2 Bcl-2 (MFI±SD) 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 ± ± 1 32 ± 2.5 CD8 + CD25-12 ± ± ± 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 ± ± ± 1 Foxp3 high Foxp3 interm. Foxp3 low CD8 + CD25-95 ± ± 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.

18 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