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Tumour cell generation of inducible regulatory T-cells in multiple myeloma is contact-dependent and antigen-presenting cell-independent.

Feyler S, Scott GB, Parrish C, Jarmin S, Evans P, Short M, McKinley K, Selby PJ, Cook G - PLoS ONE (2012)

Bottom Line: In an in vitro model, CD4(+)CD25(-)FoxP3(-) T-cells co-cultured with malignant plasma cells (primary MM cells and cell lines) induced a significant generation of CD4(+)CD25(+)FoxP3(+) inducible T(Reg) cells (tT(Reg) cells; p<0.0001), in a contact-dependent manner. tT(Reg) cells were polyclonal, demonstrated a suppressive phenotype and phenotypically, demonstrated increased FoxP3 (p = 0.0001), increased GITR (p<0.0001), increased PD1 (p = 0.003) and decreased CD62L (p = 0.007) expression compared with naturally occurring T(Reg) cells.Blocking experiments with anti-ICOS-L MoAb resulted in a significant inhibition of tT(Reg) cell generation whereas both IL-10 & TGFβ blockade did not.These features suggest that tumour generation of T(Reg) cells may contribute to evasion of immune surveillance by the host.

View Article: PubMed Central - PubMed

Affiliation: Transplant Immunology Group, Academic Department of Haematology and Oncology, University of Leeds, Leeds, United Kingdom.

ABSTRACT
Regulatory T-cells (T(Reg) cells) are increased in patients with multiple myeloma (MM). We investigated whether MM cells could generate and/or expand T(Reg) cells as a method of immuno-surveillance avoidance. In an in vitro model, CD4(+)CD25(-)FoxP3(-) T-cells co-cultured with malignant plasma cells (primary MM cells and cell lines) induced a significant generation of CD4(+)CD25(+)FoxP3(+) inducible T(Reg) cells (tT(Reg) cells; p<0.0001), in a contact-dependent manner. tT(Reg) cells were polyclonal, demonstrated a suppressive phenotype and phenotypically, demonstrated increased FoxP3 (p = 0.0001), increased GITR (p<0.0001), increased PD1 (p = 0.003) and decreased CD62L (p = 0.007) expression compared with naturally occurring T(Reg) cells. FACS-sorted tT(Reg) cells differentiated into FoxP(+)IL-17(+) and FoxP3(-)IL-17(+) CD4(+) cells upon TCR-mediated stimulation. Blocking experiments with anti-ICOS-L MoAb resulted in a significant inhibition of tT(Reg) cell generation whereas both IL-10 & TGFβ blockade did not. MM tumour cells can directly generate functional T(Reg) cells in a contact-dependent manner, mediated by ICOS/ICOS-L. These features suggest that tumour generation of T(Reg) cells may contribute to evasion of immune surveillance by the host.

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Phenotypic characteristics of tumour-induced regulatory T-cells.A. FoxP3 expression, as determined by mean fluorescence intensity (MFI) in natural TReg cells from healthy controls (Control PB, n = 43), PB from patients with MM (MM PB, n = 43) and tTReg cells (n = 15) generated in co-culture from CD4+CD25- T-cells (Kruskal-Wallis test). Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test. B. Representative histograms of naturally occurring TReg cell and tTReg cell surface expression of CD127, PD-1, GITR and CD62L. C. Summary of surface expression profiling of natural TReg cells and tTReg cells generated in co-culture, gated on FoxP3+CD25+CD4+ T-cells (n = 4), expressed as mean fluorescence intensity (MFI). Results represent all experiments, expressed as mean ± SEM and analyzed using student t-test (*p<0.003, **p<0.0001).
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pone-0035981-g002: Phenotypic characteristics of tumour-induced regulatory T-cells.A. FoxP3 expression, as determined by mean fluorescence intensity (MFI) in natural TReg cells from healthy controls (Control PB, n = 43), PB from patients with MM (MM PB, n = 43) and tTReg cells (n = 15) generated in co-culture from CD4+CD25- T-cells (Kruskal-Wallis test). Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test. B. Representative histograms of naturally occurring TReg cell and tTReg cell surface expression of CD127, PD-1, GITR and CD62L. C. Summary of surface expression profiling of natural TReg cells and tTReg cells generated in co-culture, gated on FoxP3+CD25+CD4+ T-cells (n = 4), expressed as mean fluorescence intensity (MFI). Results represent all experiments, expressed as mean ± SEM and analyzed using student t-test (*p<0.003, **p<0.0001).

Mentions: Differences in phenotype between naturally occurring and inducible TReg cells have been reported [21], [22]. We therefore sought to characterize the phenotype of tTReg cells generated in our in vitro assay compared with naturally occurring TReg cells selected from steady PB of healthy volunteers. Given the potential for heterogeneity of response between the different samples from healthy volunteers, we utilized the one HMCL to provide consistency in the in vitro model, though similar results were generated using other MM cell lines (JIM3, JJN3 & RPMI8226- data not shown). When CD4+CD25- T-cells were selected as the starting population, the level of FoxP3 expression was significantly greater than naturally occurring TReg cells either from the PB of healthy controls or patients with MM (1585±101 vs 884±67,p<0.0001, Kruskal-Wallis test; Figure 2A). Next, using a sequential gating strategy, we examined the expression of key surface markers on CD4+CD25+FoxP3+ T-cells. tTReg cells demonstrated a similar level of CD127 (p = 0.413) and CD4 (p = 0.415) expression but demonstrated significantly higher levels of CD25 (43,492±6800 vs 1896±137,p<0.0001), GITR (70±5 vs 10±3,p<0.001) and PD-1 (49.8±9 vs 5.3±0.8,p = 0.003), as illustrated in Figure 2B and C. With regards to CD62L, there was an overall lower mean fluorescence intensity (MFI) compared to naturally occurring TReg cells (88.9±0.54 vs 97.3±0.54, p = 0.008; Figure 2C), but a bi-phasic pattern of expression suggests two populations of cells, some of which demonstrated similar expression of CD62L as naturally occurring TReg cells (Figure 2C). To determine the clonality of tumour-induced TReg cells, CD4+CD25+CD127Dim T-cells were FACS sorted after 7 days of co-culture with mitomycin-C treated HMCL and DNA prepared from sorted cell populations. TCRG PCR was performed on genomic DNA derived from the tTReg cells. The spectrograph indicates multiple “spikes” representative of a polyclonal population in respect to the TCRG rearrangements, compared to a single “spike” representative of a monoclonal population (Figure S1).


Tumour cell generation of inducible regulatory T-cells in multiple myeloma is contact-dependent and antigen-presenting cell-independent.

Feyler S, Scott GB, Parrish C, Jarmin S, Evans P, Short M, McKinley K, Selby PJ, Cook G - PLoS ONE (2012)

Phenotypic characteristics of tumour-induced regulatory T-cells.A. FoxP3 expression, as determined by mean fluorescence intensity (MFI) in natural TReg cells from healthy controls (Control PB, n = 43), PB from patients with MM (MM PB, n = 43) and tTReg cells (n = 15) generated in co-culture from CD4+CD25- T-cells (Kruskal-Wallis test). Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test. B. Representative histograms of naturally occurring TReg cell and tTReg cell surface expression of CD127, PD-1, GITR and CD62L. C. Summary of surface expression profiling of natural TReg cells and tTReg cells generated in co-culture, gated on FoxP3+CD25+CD4+ T-cells (n = 4), expressed as mean fluorescence intensity (MFI). Results represent all experiments, expressed as mean ± SEM and analyzed using student t-test (*p<0.003, **p<0.0001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3362588&req=5

pone-0035981-g002: Phenotypic characteristics of tumour-induced regulatory T-cells.A. FoxP3 expression, as determined by mean fluorescence intensity (MFI) in natural TReg cells from healthy controls (Control PB, n = 43), PB from patients with MM (MM PB, n = 43) and tTReg cells (n = 15) generated in co-culture from CD4+CD25- T-cells (Kruskal-Wallis test). Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test. B. Representative histograms of naturally occurring TReg cell and tTReg cell surface expression of CD127, PD-1, GITR and CD62L. C. Summary of surface expression profiling of natural TReg cells and tTReg cells generated in co-culture, gated on FoxP3+CD25+CD4+ T-cells (n = 4), expressed as mean fluorescence intensity (MFI). Results represent all experiments, expressed as mean ± SEM and analyzed using student t-test (*p<0.003, **p<0.0001).
Mentions: Differences in phenotype between naturally occurring and inducible TReg cells have been reported [21], [22]. We therefore sought to characterize the phenotype of tTReg cells generated in our in vitro assay compared with naturally occurring TReg cells selected from steady PB of healthy volunteers. Given the potential for heterogeneity of response between the different samples from healthy volunteers, we utilized the one HMCL to provide consistency in the in vitro model, though similar results were generated using other MM cell lines (JIM3, JJN3 & RPMI8226- data not shown). When CD4+CD25- T-cells were selected as the starting population, the level of FoxP3 expression was significantly greater than naturally occurring TReg cells either from the PB of healthy controls or patients with MM (1585±101 vs 884±67,p<0.0001, Kruskal-Wallis test; Figure 2A). Next, using a sequential gating strategy, we examined the expression of key surface markers on CD4+CD25+FoxP3+ T-cells. tTReg cells demonstrated a similar level of CD127 (p = 0.413) and CD4 (p = 0.415) expression but demonstrated significantly higher levels of CD25 (43,492±6800 vs 1896±137,p<0.0001), GITR (70±5 vs 10±3,p<0.001) and PD-1 (49.8±9 vs 5.3±0.8,p = 0.003), as illustrated in Figure 2B and C. With regards to CD62L, there was an overall lower mean fluorescence intensity (MFI) compared to naturally occurring TReg cells (88.9±0.54 vs 97.3±0.54, p = 0.008; Figure 2C), but a bi-phasic pattern of expression suggests two populations of cells, some of which demonstrated similar expression of CD62L as naturally occurring TReg cells (Figure 2C). To determine the clonality of tumour-induced TReg cells, CD4+CD25+CD127Dim T-cells were FACS sorted after 7 days of co-culture with mitomycin-C treated HMCL and DNA prepared from sorted cell populations. TCRG PCR was performed on genomic DNA derived from the tTReg cells. The spectrograph indicates multiple “spikes” representative of a polyclonal population in respect to the TCRG rearrangements, compared to a single “spike” representative of a monoclonal population (Figure S1).

Bottom Line: In an in vitro model, CD4(+)CD25(-)FoxP3(-) T-cells co-cultured with malignant plasma cells (primary MM cells and cell lines) induced a significant generation of CD4(+)CD25(+)FoxP3(+) inducible T(Reg) cells (tT(Reg) cells; p<0.0001), in a contact-dependent manner. tT(Reg) cells were polyclonal, demonstrated a suppressive phenotype and phenotypically, demonstrated increased FoxP3 (p = 0.0001), increased GITR (p<0.0001), increased PD1 (p = 0.003) and decreased CD62L (p = 0.007) expression compared with naturally occurring T(Reg) cells.Blocking experiments with anti-ICOS-L MoAb resulted in a significant inhibition of tT(Reg) cell generation whereas both IL-10 & TGFβ blockade did not.These features suggest that tumour generation of T(Reg) cells may contribute to evasion of immune surveillance by the host.

View Article: PubMed Central - PubMed

Affiliation: Transplant Immunology Group, Academic Department of Haematology and Oncology, University of Leeds, Leeds, United Kingdom.

ABSTRACT
Regulatory T-cells (T(Reg) cells) are increased in patients with multiple myeloma (MM). We investigated whether MM cells could generate and/or expand T(Reg) cells as a method of immuno-surveillance avoidance. In an in vitro model, CD4(+)CD25(-)FoxP3(-) T-cells co-cultured with malignant plasma cells (primary MM cells and cell lines) induced a significant generation of CD4(+)CD25(+)FoxP3(+) inducible T(Reg) cells (tT(Reg) cells; p<0.0001), in a contact-dependent manner. tT(Reg) cells were polyclonal, demonstrated a suppressive phenotype and phenotypically, demonstrated increased FoxP3 (p = 0.0001), increased GITR (p<0.0001), increased PD1 (p = 0.003) and decreased CD62L (p = 0.007) expression compared with naturally occurring T(Reg) cells. FACS-sorted tT(Reg) cells differentiated into FoxP(+)IL-17(+) and FoxP3(-)IL-17(+) CD4(+) cells upon TCR-mediated stimulation. Blocking experiments with anti-ICOS-L MoAb resulted in a significant inhibition of tT(Reg) cell generation whereas both IL-10 & TGFβ blockade did not. MM tumour cells can directly generate functional T(Reg) cells in a contact-dependent manner, mediated by ICOS/ICOS-L. These features suggest that tumour generation of T(Reg) cells may contribute to evasion of immune surveillance by the host.

Show MeSH
Related in: MedlinePlus