Adenosine Selectively Depletes Alloreactive T Cells to Prevent GVHD While Conserving Immunity to Viruses and Leukemia

Abstract

Selective depletion (SD) of alloreactive T cells from allogeneic hematopoeitic stem cell transplants to prevent graft-versus-host disease (GVHD) without compromising immune reconstitution and antitumor responses remains a challenge. Here, we demonstrate a novel SD strategy whereby alloreacting T cells are efficiently deleted ex vivo with adenosine. SD was achieved in human leukocyte antigen (HLA) mismatched cocultures by multiple exposures to 2 mmol/l adenosine over 7 days. Adenosine depleted greater than to 90% of alloproliferating T cells in mismatched, haploidentical, and matched sibling pairs while conserving response to third-party antigens. Alloreactive CD4 and CD8 T cells were targeted for depletion while NK and B cells were preserved. Our novel approach also preserved nonalloreactive naive, central, and effector memory T-cell subsets, Tregs, and notably preserved T-cell responses against DNA viruses that contribute to transplant related mortality after allogeneic hematopoeitic stem cell transplants. Additionally, T cells recognizing leukemia-associated antigens were efficiently generated in vitro from the cell product post-SD. This study is the first to demonstrate that adenosine depletion of alloactivated T cells maintains a complete immune cell profile and recall viral responses. Expansion of tumor antigen-specific subsets postdepletion opens the possibility of generating T-cell products capable of graft-versus-tumor responses without causing GVHD.

Figure 1

Millimolar adenosine suppresses T-cell alloactivation and proliferation. (a) FACS analysis showing percent CD25⁺ T cells in culture after stimulation of healthy donor lymphocytes with HLA-mismatched recipient DC over 8 days in the presence or absence of various doses of adenosine on days 1, 2, and 5 after establishing cocultures. (b) CFSE-labeled donor lymphocytes were cocultured with HLA-mismatched recipient DCs for 10 days. Cultures were treated with 2 mmol/l adenosine on day 2 or 5 only, or days 1, 2, and 5 after establishing mMLR. Two days after the final treatment, CFSE dye dilution in T cells was determined as a measure of cell proliferation by flow cytometry. Results shown in Figure 1a and 1b are the mean of 6 HLA-mismatched donor–recipient pairs. (c) Lymphocytes from three healthy donors were stimulated with anti-CD3/CD28 beads and treated with 2 mmol/l adenosine on days 1, 2, and 5 (+Ado) or not treated (No Ado). Two days after the final treatment cultures were stained with vivid and viability was determined by flow cytometry. Results shown are representative of three donors.

Figure 2

Adenosine selectively depletes alloreactive T cells in HLA-mismatch mMLR while retaining activity against third-party APCs. Adenosine allodepleted lymphocytes (Ado) and unmanipulated lymphocytes cultured for 7 days (NT Ctrl) were stained with CFSE and tested for proliferative response against a panel of stimulators in secondary mMLR. CFSE dye dilution in T cells was determined by flow cytometry as a measure of proliferation. (a) Representative data from one donor depicting proliferation of freshly thawed lymphocytes (predepletion) and Ado-depleted lymphocytes (postdepletion) challenged against stimulator DCs for 5 days. (b) Overlayed histograms of CFSE dilution profile for representative donor shows abrogation of response against stimulator DCs. (c) Pooled data from five responder–recipient pairs, background proliferation against autologous DCs subtracted from presented values.

Figure 3

Allodepletion with adenosine preserves lymphocyte and T-cell subsets. Primary mMLR cultures were established using 12 distinct donor lymphocyte and mismatched recipient DC pairs. Cultures were treated with adenosine on day 1, 2, and 5 (Ado MLR, N = 12) or left untreated (NT MLR, N = 12). Responder lymphocytes were cultured alone (NT Ctrl, N = 6). After 7 days, the samples were harvested, washed, and phenotype was analyzed by flow cytometry. Lymphocyte subsets were identified as B cells (CD19⁺CD3^-), total NK cells (CD56⁺CD19^-), NKT cells (CD3⁺ CD56⁺), total T cells (CD3⁺CD19^-CD56^-), CD4+ T cells (CD3+CD4+), or CD8⁺ T cells (CD3+CD8+). (a, left) Total T, CD4⁺ Th, and CD8⁺ CTL frequencies. (a, right) Total NK, NKT, and B cell frequencies. (b) Gating strategy used to identify various T-cell subset denominations. CD3⁺ T cells were classified as naive (CCR7⁺CD45RO^-), central memory (CM, CCR7⁺CD45RO⁺), effector memory (EM, CCR7^-CD45RO⁺), or terminal effector (EMRA, CCR7^-CD45RO^-). (c, d) Pooled data from one experiment showing the frequency and distribution of various T-cell subsets in adenosine-treated or untreated mMLR cultures (N = 12) or responder lymphocyte alone control cultures (N = 6).

Figure 4

Tregs are not depleted by adenosine. Responder lymphocytes were cultured with no stimulation (NT Ctrl), in mMLR against mismatch stimulator DCs with adenosine treatment (Ado), or in mMLR without treatment (NT MLR). After 7 days, the cultures were analyzed to determine the frequency of CD25⁺FOXP3⁺ Tregs within CD3⁺CD4⁺ T by flow cytometry. (a) Representative dot plot analysis of sample from one donor showing the gating strategy used to determine the CD3⁺CD4⁺CD25⁺FOXP3⁺ Tregs. (b) Frequency of CD3⁺CD4⁺ CD25⁺FOXP3⁺ Tregs in NT Ctrl (N = 1), Ado (N = 3), and NT mMLR (N = 3) cultures for six donors. Each donor was depleted or cultured in mMLR against three distinct recipients.

Figure 5

T cells recognizing viral peptides persist after depletion and can be expanded through stimulation with peptide-pulsed APCs. Recognition of CMV (pp65, IE-1), EBV (EBNA1, BZLF1), and adenovirus (AD5) antigens tested by 6-hour peptide stimulation; intracellular expression of IFN-γ and TNF-α were measured by flow cytometry. (a) Lymphocytes from three CMV seropositive donors were depleted against allogeneic DCs with adenosine (Ado), cultured for an equal period with no stimulation (NT Day 7), or thawed fresh (NT Day 0) and tested against panel of viral peptide libraries. The background of IFN-γ⁺ cells against irrelevant peptide is subtracted from raw data in the presented values. (b) CMV-specific cells expanded from adenosine depleted cultures through two rounds of peptide stimulation show polyfunctional response against pp65 and IE-1 peptides.

Figure 6

Leukemia associated antigen-specific T cells were generated from allodepleted cultures. Adenosine-depleted lymphocytes from six mismatched donor–recipient pairs were stimulated twice at weekly interval with autologous DCs transduced with a lentiviral vector expressing PRAME or WT1 to generate and enrich antigen-recognizing T cells. Antigen specificity was tested against DCs transduced with the cognate antigen PRAME or WT1, or irrelevant control antigen in a 6-hour coculture assay followed by intracellular flow cytometry for cytokine production analysis. Production of IFN-γ and TNF-α in recognition of cognate antigen over background control in CD4⁺ and CD8⁺ T cell subsets is presented for (a) PRAME-specific cultures and (b) WT1-specific cultures from three donor–recipient pairs.

Figure 7

Adenosine-mediated allodepletion was highly effective in haploidentical and matched sibling mMLR. SD with adenosine was performed in mMLR coculture of “donor” haploidentical (a) and matched sibling (b) PBMCs with “recipient” DCs in three experiments. HLA serology of donors and recipients for each experiment are reported. Reactivity of allodepleted product (Ado) and untreated mMLR control (Ctrl) against donor DCs, recipient DCs, a mix of unrelated third-party DCs, and anti-CD3/CD28 beads is presented. The reported % reactivity values reflect percentage of CFSE low T cells minus background proliferation of unstimulated cultures.