Therapy of relapsed leukemia after allogeneic hematopoietic cell transplantation with T cells specific for minor histocompatibility antigens

Abstract

The adoptive transfer of donor T cells that recognize recipient minor histocompatibility antigens (mHAgs) is a potential strategy for preventing or treating leukemic relapse after allogeneic hematopoietic cell transplantation (HCT). A total of 7 patients with recurrent leukemia after major histocompatibility complex (MHC)-matched allogeneic HCT were treated with infusions of donor-derived, ex vivo-expanded CD8(+) cytotoxic T lymphocyte (CTL) clones specific for tissue-restricted recipient mHAgs. The safety of T-cell therapy, in vivo persistence of transferred CTLs, and disease response were assessed. Molecular characterization of the mHAgs recognized by CTL clones administered to 3 patients was performed to provide insight into the antileukemic activity and safety of T-cell therapy. Pulmonary toxicity of CTL infusion was seen in 3 patients, was severe in 1 patient, and correlated with the level of expression of the mHAg-encoding genes in lung tissue. Adoptively transferred CTLs persisted in the blood up to 21 days after infusion, and 5 patients achieved complete but transient remissions after therapy. The results of these studies illustrate the potential to selectively enhance graft-versus-leukemia activity by the adoptive transfer of mHAg-specific T-cell clones and the challenges for the broad application of this approach in allogeneic HCT. This study has been registered at http://clinicaltrials.gov as NCT00107354.

Figure 1

Treatment schema. Patients who relapsed after MHC-matched allogeneic HCT and elected to receive chemotherapy and T-cell therapy first underwent withdrawal of immunosuppression, followed sequentially by appropriate salvage chemotherapy, adoptive T-cell therapy with CD8⁺ mHAg-specific CTL clones, and a 14-day course of low-dose IL-2.

Figure 2

Clinical course of the 7 patients who underwent T-cell therapy. Timelines showing chronology of postrelapse treatment, disease status, histologic diagnoses of GVHD, and survival of all 7 patients who were treated for posttransplantation relapse with CD8⁺ mHAg-specific T-cell therapy.

Figure 3

Acute pulmonary toxicity in patient no. 1 associated with administration of CD8⁺ mHAg-specific CTLs. (A-B) Flow cytometric analysis of T-cell receptor expression in CTLs before infusion (A) and after recovery from bronchoalveolar lavage fluid (B) using a human TCR Vβ13-specific monoclonal antibody. (C-E) Chest radiographs taken before (C), 4 hours after (D), and 40 hours after (E) infusion of 2.25 × 10⁹ CTLs.

Figure 4

Adoptively transferred CTLs migrate to bone marrow but have limited in vivo persistence. (A) Real-time PCR with primers that specifically amplify the uniquely rearranged TCRβ CDR3 region of the mHAg-specific CTL clone 11B9-45 were used to detect and enumerate 11B9-45 CTL in the blood (■) and bone marrow (BM; ▩) of Patient no. 7 at the indicated time points before or during T-cell therapy. The level of 11B9-45 CTLs detected is expressed as a percentage of PBMCs or bone marrow mononuclear cells (BMMCs). The dose (in approximate log₁₀ scale) and timing of each of the 4 T-cell infusions administered to this patient (Tx1, Tx2, Tx3, and Tx4) are indicated. (B) Flow cytometric analysis of 11B9-45 CTLs before adoptive transfer. Cells stained with a mAb specific for the molecule indicated to the right of each histogram are indicated in red, and those stained with an isotype control antibody are indicated in green.

Figure 5

Relative expression of mHAg-encoding genes in different human tissues. Real-time PCR was used to determine the relative expression levels of the P2RX7, DPH1, and DDX3Y genes in different human tissues. Expression of the hematopoietic-specific CD45 gene was also determined to permit estimation of the extent to which each of the tissues examined was contaminated by cells of hematopoietic origin. For each gene, the relative expression level was defined as the expression level of that gene in a specific tissue compared with its expression level in male EBV-LCL. ▩ indicates expression level of each gene in the spleen and PBMCs to facilitate their comparison with the expression levels in nonhematopoietic tissues.

Figure 6

Expression of P2RX7 mRNA, protein, and mHAg in selected tissues. (A) Immunohistochemical analysis of P2RX7 expression in human lung. The width of the image is 936 microns. (B) Expression of P2RX7 (▩) and of the P2RX7-encoded mHAg recognized by CTL clone 11C6-109 (■) in patient no. 1–derived EBV-LCL, dermal fibroblasts, leukemic blasts obtained from patient no. 1 at the time of his first posttransplantation relapse (before T-cell therapy), and leukemic blasts obtained at the time of his second posttransplantation relapse, after the receipt of 6 CTL infusions. P2RX7 expression was assessed by real-time quantitative PCR, and mHAg expression was assessed by a 4-hour ⁵¹Cr release cytotoxicity assay at an E/T ratio of 10:1.

Figure 7

Differential expression of mHAg-encoding genes in pulmonary alveolar epithelial and leukemic cells. Real-time PCR analysis of P2RX7, DPH1, and DDX3Y expression in male EBV-LCL, human male pulmonary alveolar epithelial cells (HPAEpiCs), and the male-derived KG1 AML cell line was performed. The expression level of each gene was computed using the standard curve method, and the expression level in HPAEpiCs and KG1 is referenced to that observed in male EBV-LCL.