Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation

Abstract

Poor T lymphocyte reconstitution limits the use of haploidentical stem cell transplantation (SCT) because it results in a high mortality from viral infections. One approach to overcome this problem is to infuse donor T cells from which alloreactive lymphocytes have been selectively depleted, but the immunologic benefit of this approach is unknown. We have used an anti-CD25 immunotoxin to deplete alloreactive lymphocytes and have compared immune reconstitution after allodepleted donor T cells were infused at 2 dose levels into recipients of T-cell-depleted haploidentical SCT. Eight patients were treated at 10(4) cells/kg/dose, and 8 patients received 10(5) cells/kg/dose. Patients receiving 10(5) cells/kg/dose showed significantly improved T-cell recovery at 3, 4, and 5 months after SCT compared with those receiving 10(4) cells/kg/dose (P < .05). Accelerated T-cell recovery occurred as a result of expansion of the effector memory (CD45RA(-)CCR-7(-)) population (P < .05), suggesting that protective T-cell responses are likely to be long lived. T-cell-receptor signal joint excision circles (TRECs) were not detected in reconstituting T cells in dose-level 2 patients, indicating they are likely to be derived from the infused allodepleted cells. Spectratyping of the T cells at 4 months demonstrated a polyclonal Vbeta repertoire. Using tetramer and enzyme-linked immunospot (ELISPOT) assays, we have observed cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-specific responses in 4 of 6 evaluable patients at dose level 2 as early as 2 to 4 months after transplantation, whereas such responses were not observed until 6 to 12 months in dose-level 1 patients. The incidence of significant acute (2 of 16) and chronic graft-versus-host disease (GVHD; 2 of 15) was low. These data demonstrate that allodepleted donor T cells can be safely used to improve T-cell recovery after haploidentical SCT and may broaden the applicability of this approach.

Figure 1.

Kinetics of recovery of CD3⁺, CD4⁺, and CD8⁺ T, B, and NK cells after transplantation. Panel A shows CD3⁺ cells; panel B, CD4⁺ cells; panel C, CD8⁺ cells; panel D, B cells; and panel E, NK cells. Median circulating cell counts at each time point are compared between patients treated at dose levels 1 and 2. Time points at which level 2 patients had statistically significant increased counts are indicated by an asterisk.

Figure 2.

Percentage of patients achieving normal CD3, CD8, CD4, and CD19 counts, and responses to PHA stimulation. Data at 4 months are shown in panel A and 6 months in panel B.

Figure 3.

Kinetics of naive, central memory, and effector memory reconstitution after transplantation. Naive cells (CD45RA⁺CCR-7⁺) are shown in panel A; central memory (CD45RA^–CCR-7-ve) in panel B; and effector memory (CD45RA^–CCR-7^–) in panel C. Dose-level 1 patients (left column) are compared with dose-level 2 patients (right column).

Figure 4.

T-cell receptor spectratyping. (A) Boxplot of number of bands per Vβ family (1-24) at 4 and 6 months after SCT for evaluable dose levels 1 and 2 patients. Line indicates mean; box indicates 25th to 75th centile; and error bars indicate 90th and 10th percentile. (B) Representative plots of TCR Vβ repertoire in P11 (dose level 2) at 4, 6, and 12 months after SCT.

Figure 5.

Recovery of CD8 responses to viruses. (A) Recovery of CD8 responses against EBV. ▪ represents time points at which a significant (> 0.1% above isotype) tetramer-positive population was identified in the peripheral blood of recipients; □, time points at which no tetramer-positive cells were observed. The arrows indicate EBV viremia. Time points at which patient went off study for relapse (R) or mixed chimerism (MC) are shown. Crosses indicate time points at which patients died. (B) CD8 responses against CMV. Schema as in panel A. (C) Flow cytometric analysis of peripheral blood from patient P13 at 6 months after transplantation (bottom 2 panels) and his donor (top 2 panels) using CD8-FITC/tetramer-PE staining. The left panels show samples stained with the HLA-A2-NLV tetramer (pp65), and the right panels show samples stained with the HLA-B8-ELR tetramer (IE1). The percentage of CD3⁺8⁺ T cells that were tetramer positive is shown.

Figure 6.

Recovery of functional antiviral responses. (A) Functional response to EBV. The number of cells secreting interferon-γ in response to stimulation with donor EBV-LCL in ELISPOT assays are shown at varying time points after SCT. Dose-level 1 patients are shown in the top panel and level 2 patients in the bottom panel. (B) Functional response to CMV. The number of cells secreting interferon-γ in response to stimulation with donor PBMCs expressing a pp65 transgene in ELISPOT assays are shown at varying time points after SCT. Patients at dose-level 1 are shown in panel i and dose-level 2 patients in panel ii. Viral reactivations are indicated by the arrows.