Myeloid-derived suppressor cells promote cross-tolerance in B-cell lymphoma by expanding regulatory T cells

Abstract

Tumor-induced T-cell tolerance is a major mechanism that facilitates tumor progression and limits the efficacy of immune therapeutic interventions. Regulatory T cells (Treg) play a central role in the induction of tolerance to tumor antigens, yet the precise mechanisms regulating its induction in vivo remain to be elucidated. Using the A20 B-cell lymphoma model, here we identify myeloid-derived suppressor cells (MDSC) as the tolerogenic antigen presenting cells capable of antigen uptake and presentation to tumor-specific Tregs. MDSC-mediated Treg induction requires arginase but is transforming growth factor-beta independent. In vitro and in vivo inhibition of MDSC function, respectively, with NOHA or sildenafil abrogates Treg proliferation and tumor-induced tolerance in antigen-specific T cells. These findings establish a role for MDSCs in antigen-specific tolerance induction through preferential antigen uptake mediating the recruitment and expansion of Tregs. Furthermore, therapeutic interventions, such as in vivo phosphodiesterase 5-inhibition, which effectively abrogate the immunosuppressive role of MDSCs and reduce Treg numbers, may play a critical role in delaying and/or reversing tolerance induction.

Figure 1. Phenotypic analysis of APCs in lymphoma

Splenocytes from CD45.1^+/+ BALB/c mice challenged 28 days before with 10⁶ CD45.2⁺ A20-HA/GFP cells or with CD45.2⁺ A20-HA as control, were stained with anti-CD45.2-PerCP, anti-CD11b-PE and anti-CD11c-APC antibodies and analyzed by FACS. A) Gating strategies: a first gate (Live) was drawn on the Forward vs side scatter plot to exclude aggregate and cellular debris, while a second gate (APCs: Antigen Presenting Cells) was designed on the GFP⁺ CD45.2^- population. B) CD11b and CD11c population were determined by gating in Live (B, left dot plot) or in Llive and APC (B right dot plot). C) Alternatively splenocytes were labeled with anti-CD45.2-PerCP, anti-CD11b-APC antibodies and with PE conjugated antibodies against the indicated marker. The histograms are gated on the hierarchical gate CD11b⁺ and APCs and Live. Data represent 3 pooled spleens representative of other 2 experiments. 10⁵ events were acquired for analysis.

Figure 2. Molecular properties of CD11b⁺ cells isolated from lymphoma-bearing mice

Mice were injected intravenously with 10⁶ A20-HA cells or with PBS on day 0. On day 28 splenic CD11b⁺ cells were magnetically purified. A) An aliquot of CD11b⁺ cells (10⁵ cells) was admixed with HA-specific, thy1.1⁺, CFSE labeled, magnetically purified CD4⁺ or CD8⁺ cells (10⁵) in the presence of Thy1.2 Balb/c splenocytes as feeder (10⁶) with or without relevant peptide. After 5 days, cell proliferation was determined as CFSE dilution by FACS analysis by gating on the clonotypic population. Data are normalized on the controls (proliferation of T cells cultured with CD11b⁺ cells isolated from naïve mice). B) Aliquots of CD11b⁺ were frozen and later tested for arginase or plated to determine TGF-β secretion or NO production as described in the materials and methods. C) The proliferation was repeated as described in (A) in the presence of NOHA (an arginase inhibitor), L-NMMA (a NOS2 inhibitor), anti-TGF-β neutralizing antibody, or with different combinations of these molecules. Data derived from one experiment representative of two.

Figure 3. FOXP3⁺ CD4 T cells are increased after co-cultured with MDSCs

A) CD11b⁺ cells were isolated from naïve or A20-HA tumor-bearing mice. 10⁵ cells were then co-cultured with 10⁵ HA-specific, magnetically purified, CFSE labeled, Thy1.1⁺CD4⁺ T cells and with 10⁶ Thy1.1^-/- BALB/c splenocytes in the presence or absence of the relevant peptide. After 5 days the cultures were labeled with anti-CD4 and anti-thy1.1, permeabilized and then labeled with anti-FOXP3. The dot plots are gated on the clonotypic T cell population. B) Results from 3 replicate wells are represented as the histogram mean +/-SD of FOXP3⁺CFSE^low cells gated on the CD4⁺/Thy1.1⁺ population. C) Either high purity CD11b^high,CD11c^low MDSC or high purity CD11b^low,CD11c^high MHC class II^high dendritic cells were sorted using a FacsAria cell sorter from spleens of mice challenged with A20WT or A20HA 28 days prior. MDSCs (10⁵) or DCs (10⁵) were cultured with CFSE-labeled, HA-specific, Thy1.1^+/+ CD4⁺ T cells (10⁵) and Thy1.2^+/+ Balb/c splenocytes (10⁶) as feeder cells. The cultures were analyzed after 3 days by flow cytometry as described in (A). The proliferation of clonotypic FOXP3⁺ T cells (left panel) or clonotypic FOXP3^neg T cells (right panel) was analyzed using Cell Diva and FCS software. C) The same experiment described in (A) was conducted in the presence of the inhibitors described in Fig2. Data derived from triplicate wells of 1 experiment representative of a total of 3 experiments.

Figure 4. Expanded Treg are derived from a preexisting regulatory pool and not from the conversion of FOXP3^neg CD4⁺ T cells

A) HA-specific CD4⁺/CD25⁺ T cells (10⁴) isolated from Thy1.1^+/+ 6.5 mice and CD4⁺CD25^- T cells purified from Thy1.1^+/-/Thy/1.2^+/- 6.5 mice (10⁵) were cultured with Thy1.2^+/+ Balb/c splenocytes (10⁶) and with splenic CD11b⁺ cells (10⁵) magnetically purified from the spleens of mice injected with PBS, A20HA or A20WT 28 days prior. No exogenous peptide was added to the culture. Five days later cells were labeled with anti thy1.1, anti thy1.2 and FOXP3 antibodies and analyzed by FACS. Average +/- SD of triplicate wells was analyzed by gating either on the thy1⁺thy1.2^- population (black bar) or on the Thy1.1⁺/Thy1.2⁺ population (gray bar) from the mixture culture (upper panel) or on CD25-depleted, HA-specific, CD4⁺ T cells cultured with the same CD11b⁺ cells (lower panel-effector alone). B) The experiment was repeated with CFSE labeling of T cells. Proliferation was determined by CFSE dilution on either the CD4⁺ Thy1.1⁺/Thy1.2^- “Treg” population (black bars) or on the Thy1.1⁺/Thy1.2⁺ “effector” population (gray bars). C) HA-specific CD4⁺CD25⁺T cells isolated from Thy1.1^+/+ Thy1.2^-/-6.5 mice were admixed (1:10 ratio) with CD4⁺CD25^- T cells sorted from Thy1.1^+/-/Thy/1.2^+/- 6.5 mice (left panel). This mixture was CFSE labeled and injected alone or with purified CD11b⁺ cells from mice injected with A20-HA or A20WT 28 days prior in a 1:1 ratio into Thy1.2 Balb/c mice. Splenocytes were examined three days later for Thy1.1 and Thy1.2 expression (right panel). Adoptively transferred “Tregs” can be identified as the Thy1.1⁺/Thy1.2^- population while “effector” T cells by co-expression of Thy1.1 and Thy1.2. Proliferation of each population was determined by CFSE dilution. (D) 10⁶ splenocytes were acquired and analyzed. Results are expressed as the mean ± SD of 3 mice.

Figure 5. IL4Rα expression on CD11b⁺ cells correlates with tumor progression and can be down-modulated by sildenafil

Mice were injected on day 0 with 10⁶ A20-HA cells and either treated with sildenafil or left untreated. At the indicated time points, mice were sacrificed and splenocytes analyzed by FACS for (A) IgG_2a expression - as a measure of tumor burden, or (B, C) for the co-expression CD11b, Gr1 and IL4Rα to determine MDSC expansion.

Figure 6. In vivo sildenafil administration reduces Treg proliferation and prevents T cell anergy

A) Experimental schema. Mice were injected with A20-HA (10⁶cells) or PBS on day 0. All the mice received 2.5×10⁶ HA-specific, Thy1.1^+/+, CD4 purified T cells on day 10 and half were then administered sildenafil in the drinking water. On day 25 half of the mice in each group were vaccinated with VaccHA (10⁷ PFU) and sacrificed 5 days later. (B) clonal expansion and (C) FOXP3 expression was evaluated by FACS analysis. (D) The experiment was repeated as described above with CFSE labeled clonotypic T cells. CD4⁺ T cells were purified on day 30 and CD4⁺/Thy1.1⁺/CFSE^low cells were further purified by cell sorting. To evaluate their suppressive activity, 3×10⁴ CFSE^low cells were co-cultured with naïve HA-specific T cells in the presence of the relevant peptide and irradiated BALB/c splenocytes as feeder cells. IFN-γ production was evaluated 48h later by ELISA.