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

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.

Figure 1. Regulatory T-cell induction by Myeloma tumour cells.

A. The expansion of FoxP3 ⁺CD25⁺CD4⁺ natural T_Reg cells, expressed as a percentage of CD4⁺T-cells. T_Reg cells were enumerated in the PB of healthy donors, after 7 days in CM and 7 days co-cultured with mitomycin C-treated U266B cells (n = 4). Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test. B. Expansion of nT_Reg cells when co-culture with HMCL results from cell division, illustrated by a representative flow cytometry plot of CFSE dilution. C. The generation of FoxP3 ⁺CD25⁺CD4⁺ T-cells, expressed as a percentage of CD4⁺T-cells, in a co-culture assay with mitomycin C-treated U266B cells (n = 6) with varying starting populations: PB MNC, PBL CD25 depleted and CD4⁺CD25^- T-cells. Results demonstrate that increased generation of tumour-induced regulatory T-cells (tT_Reg cells) is seen with increasing purity of the seeded population. Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test. D. Representative flow cytometry plots demonstrating the generation of FoxP3 ⁺CD25⁺CD4⁺ T-cells from CD4⁺CD25^- T-cells through cell division of de novo generated FoxP3 ⁺ T-cells in a 7 day co-culture assay with mitomycin C-treated U266B cells. E. The generation of FoxP3 ⁺CD25⁺CD4⁺ T-cells, expressed as a percentage of CD4⁺T-cells, in a co-culture assay of CD4⁺CD25⁻ T-cells (n = 10) with mitomycin C-treated MM cell lines (U266B, JJN3, JIM3 & KMS11), an erythro-leukaemia cell line (K562) and non-heamatopoietic cell lines (Mel888 & HeLa). Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test (**p<0.001, *p<0.01). F. The generation of FoxP3 ⁺CD25⁺CD4⁺ T-cells, expressed as a percentage of CD4⁺T-cells, in a co-culture assay with fresh BM-derived myeloma plasma cells from patient samples (n = 7). Results demonstrate that increased generation of tumour-induced regulatory T-cells (tT_Reg cells) is seen with primary myeloma cells. Results represent all experiments, expressed as mean ± SEM and analyzed using a 1-way ANOVA and student t-test.

Figure 2. Phenotypic characteristics of tumour-induced regulatory T-cells.

A. FoxP3 expression, as determined by mean fluorescence intensity (MFI) in natural T_Reg cells from healthy controls (Control PB, n = 43), PB from patients with MM (MM PB, n = 43) and tT_Reg 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 T_Reg cell and tT_Reg cell surface expression of CD127, PD-1, GITR and CD62L. C. Summary of surface expression profiling of natural T_Reg cells and tT_Reg 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).

Figure 3. Functional characteristics of tumour-induced regulatory T-cells.

A. Suppression of anti-CD3/anti-CD28-induced autologous T-cell proliferation by tumour-generated and naturally occurring T_Reg cells (n = 3), as determined by tritiated thymine incorporation. Results expressed as counts per minute (cpm)± SEM representing assays performed in triplicate. Key: Unstim – resting CD4⁺CD25^- T cells, Stim – CD3/CD28 stimulated CD4⁺CD25^- T-cells, 4∶1 etc – ratio of stimulated autologous T-cells to T_Reg cells. B. The generation of IL-10 in co-cultures of CD25^-CD4⁺ sorted T-cells and HMCL, compared with HMCl alone and culture medium (n = 6, p = 0.0004). Results represent all experiments, expressed as mean±SEM and analyzed using student t-test. C. IL-10 production by tT_Reg cells after 7 days of co-cultures of CD25^-CD4⁺ sorted T-cells and HMCL. Results represent all experiments, expressed as mean±SEM (n = 3) and analyzed using student t-test. D. IL-10 production by tT_Reg cells after 7 days of co-cultures of CD25^-CD4⁺ sorted T-cells and HMCL. Results represent all experiments, expressed as mean±SEM (n = 3) and analyzed using student t-test. E. Representative flow cytometry plots demonstrating the generation of IFNγ⁺ FoxP3 ⁺CD25⁺CD4⁺ T-cells from CD4⁺CD25^- T-cells in a 7 day co-culture assay with mitomycin C-treated U266B cells. F. The proportion of IFNγ-producing FoxP3 ⁺CD25⁺CD4⁺ T-cells detectable in the peripheral blood of age-matched controls (n = 15), patients with MM (n = 15) and tTReg cells generated in vitro after 7 days of co-cultures of CD25^-CD4⁺ sorted T-cells and HMCL (n = 3). Histograms represent IFNγ production by cells gated on FoxP3/CD25/CD4 positive staing. Results expressed as mean±SEM.

Figure 4. Natural and tumour-induced regulatory T-cell plasticity.

A. The generation of IL-17 in co-culture supernatants of CD25^-CD4⁺ sorted T-cells with HMCL, compared with HMCl alone and culture medium (n = 3). Results represent all experiments, expressed as mean±SEM. B. Representative dot-plots of IL-17 producing cells generated from re-stimulation of sorted tumour-generated and naturally occurring T_Reg cells after 5 days of re-stimulation. C. The proportion of IL-17 producing cells generated from re-stimulation of sorted tumour-generated and naturally occurring T_Reg cells, expressing FoxP3 after 5 days of re-stimulation, expressed as a percentage of CD4⁺ T-cells. Results expressed as mean±SEM.

Figure 5. In vitro mechanisms of tumour regulatory T-cell induction.

A. The generation of FoxP3 ⁺CD25⁺CD4⁺ tT_Reg cells from CD4⁺CD25^- T-cells, expressed as a percentage of CD4⁺T-cells, in a co-culture assay with mitomycin C-treated U266 cells with and without transwell inserts (n = 7). Results represent all experiments, represented as mean±SEM and analysed using a student t-Test. B. Surface expression of HLA-DR, ICOSL (CD275) and TGFβ by human myeloma cell lines. C. Inhibition of tT_Reg cells generation from CD4⁺CD25^- T-cells by co-culture with HMCL (n = 3) through blockade of TGFβ and IL-10 using monoclonal antibodies and Latency-associated Peptide (LAP). Results represent all experiments, expressed as mean (±SEM). D. Surface expression of ICOS by T_Reg cells, presented as both percentage expression of CD4⁺CD25⁺ FoxP3 ⁺ cells and representative dot plots. Results represent all experiments, expressed as mean ±SEM (n = 3) and analysed using a student t-test. E. Inhibition of tT_Reg cells generation from CD4⁺CD25^- T-cells by co-culture with HMCL through blockade of anti-ICOS-L (αICOS 1, 10, 100 µM) monoclonal antibody (n = 6), expressed as percentage of CD4⁺ T-cells and percent inhibition of tT_Reg cell generation. Results represent all experiments, illustrated as median with maximum and minimum values and analysed using a student t-test.