Molecular and functional characterization of allogantigen-specific anergic T cells suitable for cell therapy

Abstract

Background: CD4(+) regulatory T cells are a specialized subset of T cells that actively control immune responses. Several experimental protocols have been used to expand natural regulatory T cells and to generate adaptive type 1 regulatory T cells for regulatory T-cell-based therapies.

Design and methods: The ability of exogenous recombinant human interleukin-10 to induce alloantigen-specific anergy in T cells was investigated and compared to that of interleukin-10 derived from tolerogenic dendritic cells, in mixed lymphocyte cultures. A detailed characterization of the effector functions of the resulting anergized T cells is reported.

Results: Interleukin-10, whether exogenous or derived from tolerogenic dendritic cells, induces a population of alloantigen-specific T cells (interleukin-10-anergized T cells) containing type 1 regulatory T cells, which are anergic and actively suppress alloantigen-specific effector T cells present within the mixed population. Interleukin-10-induced anergy is transforming growth factor-β independent, and is associated with a decreased frequency of alloantigen-specific cytotoxic T lymphocyte precursors, but interleukin-10-anergized T cells are still responsive to third-party, bacterial, and viral antigens. Tolerogenic dendritic cells are more powerful than exogenous interleukin-10 in generating type 1 regulatory T-cell precursors, and are also effective in the context of HLA-matched donors.

Conclusions: Based on these studies, we have developed an efficient and reproducible in vitro method to generate antigen-specific type 1 regulatory T-cell precursors starting from total peripheral blood cells with minimal cell manipulation and suitable for generating type 1 regulatory T cells for regulatory T-cell-based therapies.

Figure 1.

IL-10 inhibits primary allo-responses and induces anergy associated with a decreased frequency of antigen-specific CTL precursors. (A) PBMC were stimulated with allogeneic monocytes (CD3-depleted cells) in the absence (white columns, MLR) or presence of IL-10 (black columns, MLR/10). Proliferative responses were evaluated after 4 days of culture by adding ³H-thymidine for an additional 16 h. (B) PBMC were stimulated with allogeneic monocytes without (white columns, MLR) or with IL-10 (black columns, MLR/10) for 10 days. At the end of culture, T cells were collected and tested for their ability to respond to the same allogeneic monocytes used in the primary stimulation. Proliferative responses were evaluated after 48 h of culture by adding ³H-thymidine for an additional 16 h. Results represent the mean±SD of triplicate experiments. Three haploidentical donors (#1, #2, #3) defined as ≥2 HLA-A, B, C, DRB1 disparity, and three mismatched donors (#4, #5, #6) defined as totally mismatched pairs, are shown. Numbers indicate % of inhibition of proliferation in the primary stimulation (A) and % of anergy in the secondary stimulation (B), calculated as follows: 100-[(MLR cpm-MLR/10 cpm)/MLR*100]. C. PBMC were stimulated with CD3-depleted cells in the absence (MLR) or presence of IL-10 (MLR/10) for 10 days. After cultures T cells were incubated with haploidentical human skin biopsies. Hematoxylin and eosin staining of human skin biopsies incubated with medium (control skin), control cultures (skin + MLR cells), and IL-10-anergized cultures (skin + MLR/10 cells) are presented. One representative experiment out of three is shown. (D) PBMC were stimulated with allogeneic monocytes in the absence (MLR, black dots) or in the presence of IL-10 (MLR/10, white dots) for 10 days. After culture, T cells were collected and specific lysis of allogeneic stimulators used in the priming was evaluated. Cultures were tested in escalating concentrations in 20 replicates. The cut-off line of positive lysis was based on lytic activity against autologous cells ±3 SD. Representative data for one mismatched pair out of 20 tested and one haploidentical pair out of 10 tested are shown.

Figure 2.

IL-10 inhibits flu-specific T-cell responses without deleting antigen-specific effector T cells. (A) CD4^-CD19^- PBMC isolated from a HLA-A2 typed healthy donor were stimulated with MP.58-66 peptide, in the absence (MLP) or presence of IL-10 (MLP/IL-10). After two rounds of stimulation, MLP and MLP/10 cells were re-challenged with T2 cells pulsed with MP.58-66 peptide and MP.58-66-specific lysis was measured. Lysis against unpulsed T2 targets was subtracted. One representative experiment out of three is shown. Results represent the mean±SD of triplicate experiments. (B) In parallel, MLP and MLP/10 cells were re-challenged with T2 cells pulsed with MP.58-66 peptide and IFN-γ was quantified in culture supernatants by ELISA. Results from each donor tested are shown. Results represent the mean±SD of triplicate experiments. The % inhibition of IFN-γ release in the presence of IL-10 relative to control is presented. (C) MLP and MLP/10 cells were stained with TCR-Vβ17-specific monoclonal antibody. Data are expressed as % of CD8⁺Vβ17⁺ T cells. Results from each of the seven donors tested are shown. (D) MLP and MLP/10 cells were tested in the tetramer-binding assay. Data are expressed as % of CD8⁺ T cells binding A2/MP.58-66 tetramers. Results from each of the three donors tested are shown.

Figure 3.

IL-10-anergized cultures preserve the ability to proliferate towards nominal or third-party antigens. PBMC were stimulated with allogeneic monocytes without (MLR) or with IL-10 (MLR/10) for 10 days. (A) After culture, MLR (white bars) and MLR/10 (black bars) cells were stimulated with autologous monocytes in the presence of tetanus toxoid, Candida albicans, cytomegalovirus (CMV), and 3^rd-party monocytes. Proliferative responses were evaluated after 4 days of culture by the addition of ³H-thymidine for an additional 16 h. Results are from one mismatched (defined as totally mismatched) and one haploidentical (defined as 2 HLA-A, B, C, DRB1 disparity) pair. Results represent the mean±SD of triplicate experiments. (B) Alternatively, the frequency of cytotoxic T cells specific for the EBV-LCL detected in MLR (black dots) and in MLR/10 (white dots) cultures was evaluated. The assay was performed in 20 replicates for escalating concentrations of cells. The cut-off line between positive and negative values was based on lytic activity obtained against autologous PHA-blasts. Results from one donor out of four tested are shown.

Figure 4.

DC-10 induce T-cell hypo-responsiveness and T-cell anergy associated with a high frequency of IL-10-producing T cells. (A) PBMC were stimulated with allogeneic mDC (white columns, MLR/mDC) or with DC-10 (black columns, MLR/DC-10). Proliferative responses were evaluated after 4 days of culture by adding ³H-thymidine for an additional 16 h. Results represent the mean±SD of triplicate experiments. (B) PBMC were stimulated with allogeneic mDC (white columns, MLR/mDC) or with DC-10 (black columns, MLR/DC-10) for 10 days. At the end of culture, T cells were collected and restimulated with the mDC from the same donor used in the primary stimulation. Proliferative responses were evaluated after 48 h of culture, by adding ³H-thymidine for 16 h. Three mismatched donors (#1, #2, #3) (defined as totally mismatched), two haploidentical donors (#4, #5) (defined as ≥2 HLA-A, B, C, DRB1 disparity) and three MUD donors (#7; one mismatch HLA-Cw02 versus HLA-Cw15, #8; two mismatches HLA-DPB1 0401 versus HLA-DPB1 0402, 0501, #9; one mismatch HLA-Cw03 versus HLA-Cw04) pairs are shown. Results represent the mean±SD of triplicate experiments. Numbers indicate % inhibition of proliferation (A) and of anergy in terms of proliferation (B) calculated as follows: 100-[(MLR/mDC - MLR/DC-10) / MLR/mDC*100]. (C) PBMC were stimulated with allogeneic mDC (white columns, MLR/mDC), DC-10 (black columns, MLR/DC-10) or with CD3-depleted cells in the absence (bright gray columns, MLR) or presence of IL-10 (gray columns, MLR/10) for 10 days. At the end of culture, T cells were collected and restimulated with the mDC or CD3-depleted cells from the same donor used in the primary stimulation. Specific IL-10-producing T cells present in the cultures were counted by ELISPOT. Two different experiments are shown. Results represent the mean±SEM of triplicate experiments.