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Immunological Outcome in Haploidentical-HSC Transplanted Patients Treated With IL-10-Anergized Donor T Cells

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Immunological Outcome in Haploidentical-HSC Transplanted Patients Treated With IL-10-Anergized Donor T Cells

Rosa Bacchetta et al. Front Immunol.

Abstract

T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent disease recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSCT (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10-anergized donor T cells (IL-10-DLI) contained T regulatory type 1 (Tr1) cells specific for the host alloantigens, limiting donor-vs.-host-reactivity, and memory T cells able to respond to pathogens. IL-10-DLI were infused in 12 patients with the goal of improving immune reconstitution after haplo-HSCT without increasing the risk of graft-versus-host-disease (GvHD). IL-10-DLI led to fast immune reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vβ repertoire and T-cell functions progressively normalized after IL-10-DLI. These four patients are alive, in complete disease remission and immunosuppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted (IR) patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1-specific biomarkers in vivo. Gene-expression profiles of IR patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders.

Keywords: IL-10; T regulatory type 1 cells; cell therapy; haploidentical; hematopoietic stem cell transplantation; tolerance.

Figures

Figure 1
Figure 1
Patients’ distribution and overall outcome of patients enrolled in ALT-TEN trial. IL-10-DLI: IL-10-anergized donor T cells; immune reconstituted patients: CD3+ T cells >100/μl.
Figure 2
Figure 2
Immune reconstitution in long-term-surviving IL-10-DLI patients. (Pt 6, 7, 9, and 11). (A) Kinetics of recovery of peripheral lymphocyte subsets (absolute number), at different time-points (d, day; y, year) after infusion of IL-10-anergized donor T cells (IL-10-DLI). For patient 9, who received two subsequent IL-10-DLI, timing is calculated from the second IL-10-DLI. Reference values for healthy controls (HC) are: total lymphocytes 1000–4800/μl, CD3+ T cells 781–2664/μl, CD3+CD4+ T cells 397–1762/μl, CD3+CD8+ T cells 224–1762/μl, CD16+CD56+ NK-cells 56–690/μl, CD19+ B cells 57–710/μl. Gray bars indicate the time-frame of immunosuppression (IS). Infective episodes (febrile episodes of bacterial, fungal or unknown origin) and viral (cytomegalovirus, Varicella-Zoster virus, Herpes-Simplex virus, Hepatitis B virus) reactivations detected by molecular analysis prior to the appearance of clinical manifestations are indicated (filled and empty diamonds, respectively). (B) Relative distribution of naïve and memory in gated CD3+ T cells. E, effector T cells (CD62LCD45RA+); EM, effector-memory T cells (CD62LCD45RA); CM, central memory T cells (CD62L+CD45RA); N, naïve (CD62L+CD45RA+) T cells. HC, n = 20. (C) Relative distribution of B-cell subsets, according to the expression of CD24 and CD38 in gated CD19+ B-cells. T, transitional B cells (CD24brightCD38bright); N, naïve/mature B cells (CD24dullCD38+); M, memory B cells (CD24brightCD38). HC, n = 3.
Figure 3
Figure 3
TcLandscape® prior and at different time-points after IL-10-DLI treatment in haplo-HSC transplanted patients. T cell receptor (TCR) repertoires of the donor’s PBMC, IL-10-anergized donor T cells (IL-10-DLI) prior to infusion, and patient’s PBMC before and at different time-points post-IL-10-DLI, in the presence and absence of GvHD and immunosuppression (IS). (A) One representative patient (pt 11) out of four studied is shown. Four-dimensional graphs of the different Vβ families are shown. X axis, Vβ gene family; y axis, length of the complementarity- determining-region-3 (CDR3); z axis, amount of Vβ gene normalized by hypoxanthine-guanine phosphoribosyl transferase; color code, percentage of perturbation compared to normal Gaussian distribution; d, days; y, years. (B) TCR repertoire analysis. Evolution of Vβ gene-expression of each patient by Principal Component Analysis (PCA). PCA allows projection of the different time-points in a factorial space defined by axes, named “components,” which are linear combinations of optimally weighted observed variables. The distance between two time-points is related to their TcLandscape similarity. For each patient, the percentages of each Vβ family for all recipient time-points, were independently analyzed by PCA. The “itinerary” taken by the TCR repertoire of the patient during T-cell reconstitution is shown by a dashed curve and arrows. Pt: patient; IL-10-DLI: IL-10-anergized donor T cells infused into the patients; d: days; y: years.
Figure 4
Figure 4
Immune functions in long-term surviving IL-10-DLI treated patients. (A) In vitro proliferative responses of PBMC to mitogens and specific antigens. Data are expressed as counts per minute (cpm) and are normalized for absolute number of lymphocytes: cpm obtained in response to addition of exogenous IL-2 are normalized to total lymphocyte counts. Cpm obtained in response to anti-CD3/CD28 (aCD3/CD28) monoclonal antibodies and other stimuli, phytohemagglutinin (PHA), and to the viral antigens Herpes-Simplex virus (HSV) and cytomegalovirus (CMV), are normalized to the number of CD3+ T cells. Asterisks indicate proliferative responses of haploidentical donor PBMC before transplant. Pt: patient; d: days; y: years. (B) Frequency of CMV- and HSV-specific T cells producing interferon (IFN)-γ in patients’ PBMC. Bars represent the number of IFN-γ producing T cells specific for CMV, HSV and PMA+ ionomycin (PMA/iono) in peripheral blood of patients (patients 6, 7, 9, and 11) tested 4, 3.5, 2.7, and 2.5 years after IL-10-DLI, respectively. Healthy controls (HC) were tested in parallel. Pt: patient.
Figure 5
Figure 5
Response to host allo-Ags and biomarkers of tolerance. (A) Proliferative responses of donor-derived PBMC, toward host-monocytes before transplantation (pre). Proliferative responses of patient-derived PBMC (4, 3.5, 2.5, and 2.5 years, post-IL-10-DLI for pt 6, 7, 9, and 11, respectively) toward host-derived or third-party mature dendritic cells (post). (B) Intracytoplasmic granzyme-B (GZ-B) and IL-10 on gated CD4+ T cells from total PBMC of patients 6, 7, 9, and 11, tested 175 days, 2.8, 3, and 5 years; 180 days, 1.5, 2.5, 4, 5, and 7 years; 180 days, 1.5, 2, 2.7, 4.2, 5, and 6 years; 190 days, 1 year, 3.5, 4, 4.2, and 6 years after IL-10-DLI, respectively, and of healthy controls (HC) (HC: n = 10). Mean ± SD of the percentage of IL-10+ T cells of GzB+ T cells and of IL-10+GzB+ T cells in all time-points analyzed are shown, Differences were evaluated by means of the non-parametric Mann-Whitney U test. P-values < 0.05 were considered significant. (C) Surface CD49b and LAG-3 on gated CD4+ T cells from total PBMC of patients 6, 7, 9, and 11, tested 175 days, 2.8, 3, and 5 years; 180 days, 1.5, 2.5, 4, 5, and 7 years; 180 days, 1.5, 2, 2.7, 4.2, 5, and 6 years; 190 days, 1 year, 3.5, 4, 4.2, and 6 years after IL-10-DLI, respectively, and of HC (HC: n = 5), and of donor PBMC of pt 7 and 9 (Donor pre-Tx). Mean ± SD of the percentages of CD49b+LAG-3+ T cells at indicated time-points are shown. Differences were evaluated by means of the non-parametric Mann-Whitney U test. P-values < 0.05 were considered significant. cpm: counts per minutes of Tritiated Thymidine incorporation.
Figure 6
Figure 6
Gene-expression profile analysis of three long-term follow-up patients. (A) Matrix view of gene-expression data (heat map) showing genes with significantly increased (violet vertical bar) and decreased (pink vertical bar) expression levels in samples evaluated at different time-points post-IL-10-DLI (as indicated in days or years) of patient 7, patient 9, and patient 11 compared to three samples from healthy controls (HC). Data represent quantiles normalized and median-centered signal intensity data in log 2 space. Red and green indicate upregulation and downregulation, respectively, relative to the reporter-wise median. (B) Gene-expression analysis of the ten most discriminatory biomarkers of tolerance identified within the Indices of Tolerance (IOT) study in patients 7, 9, and 11. Pairwise Pearson correlation coefficients comparing the medians of expression in each IOT patient group (Tol-DF: tolerant drug-free; s-CNI: stably immunosuppressed by calcineurin inhibitors (CNI); s-nCNI: stable under non-CNI; s-LP: stable under low dose prednisolone; HC: healthy controls) with the expression in each separate time-point sample from patients 7, 9, and 11 are shown. The expression of the most discriminatory IOT biomarkers in IL-10-DLI treated patients progressively became super-imposable to that in Tol-DF patients and HC. The gray bars below each graph indicate immunosuppressive treatment.

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