Development of tumor-reactive T cells after nonmyeloablative allogeneic hematopoietic stem cell transplant for chronic lymphocytic leukemia

Abstract

Purpose: Allogeneic nonmyeloablative hematopoietic stem cell transplant (NM-HSCT) can result in durable remission of chronic lymphocytic leukemia (CLL). It is thought that the efficacy of NM-HSCT is mediated by recognition of tumor cells by T cells in the donor stem cell graft. We evaluated the development of CTLs specific for CLL after NM-HSCT to determine if their presence correlated with antitumor efficacy.

Experimental design: Peripheral blood mononuclear cells obtained from 12 transplant recipients at intervals after NM-HSCT were stimulated in vitro with CLL cells. Polyclonal T-cell lines and CD8(+) T-cell clones were derived from these cultures and evaluated for lysis of donor and recipient target cells including CLL. The presence and specificity of responses was correlated with clinical outcomes.

Results: Eight of the 12 patients achieved remission or a major antitumor response and all 8 developed CD8(+) and CD4(+) T cells specific for antigens expressed by CLL. A clonal analysis of the CD8(+) T-cell response identified T cells specific for multiple minor histocompatibility (H) antigens expressed on CLL in six of the responding patients. A significant fraction of the CD8(+) T-cell response in some patients was also directed against nonshared tumor-specific antigens. By contrast, CLL-reactive T cells were not detected in the four patients who had persistent CLL after NM-HSCT, despite the development of graft-versus-host disease.

Conclusions: The development of a diverse T-cell response specific for minor H and tumor-associated antigens expressed by CLL predicts an effective graft-versus-leukemia response after NM-HSCT.

Figure 1. Cytotoxicity of T cell lines generated from NM-HSCT recipients by stimulation with recipient CD40-CLL

PBMC were obtained from recipients at the designated days after NM-HSCT, stimulated twice one week apart with γ-irradiated recipient CD40-CLL, and tested in a cytotoxicity assay against Cr⁵¹-labeled recipient CD40-CLL (■), recipient B-LCL () and donor B-LCL (□). Data is shown for an effector to target ratio of 30:1 or 40:1. A. Reactivity of T cell lines generated from patients who had a major antitumor response after NM-HSCT. B. Reactivity of T cell lines generated from patients who had persistent or progressive disease after NM-HSCT.

Figure 2. CD40-CLL from patients who fail to respond to nonmyeloablative HSCT are effective antigen presenting cells

A–B PBMC from an unrelated HLA disparate individual (JKW) were stimulated in a mixed lymphocyte culture twice one week apart with γ-irradiated CD40-CLL derived from a responding patient (UPN 22388) as a control (A), and withγ-irradiated CD40-CLL from each of the 4 transplant recipients who failed to achieve a CR after transplant (B). The T cell lines were then assayed for cytotoxic activity against autologous (JKW) B-LCL, recipient CD40-CLL and recipient B-LCL if available at effector to target ratios of 1:1 – 100:1. C. CLL cells and B-LCL from an HLA-A2⁺ male patients who had progressive CLL after NM-HSCT (28196) or who responded to NM-HSCT (22388) were tested for the ability to stimulate an SCMY-specific T cell clone (CTL) to produce IFN-γ. T cells and stimulator cells were co-cultured at a ratio of 3:1 for 24 hours, supernatants were collected and IFN-γ was measured by ELISA.

Figure 3. CLL-reactive CD4⁺ and CD8⁺ T cells that produce IFN-γ and cytolytic CD8⁺ T cells are elicited after NM-HSCT in responding patients

A, B T cell lines from two representative patients (UPN 22388 and UPN 9661) were stimulated with recipient CD40-CLL (top panels) and B-LCL (middle panels), and with donor B-LCL (lower panels) and stained with PE-conjugated IFN-γ detection reagent and either FITC-conjugated anti-CD4 or anti-CD8 monoclonal antibodies. C, D. CD4⁺ and CD8⁺ T cells were isolated by IFN-γ⁺ capture after stimulation with recipient CD40-CLL, expanded with anti-CD3 monoclonal antibody and tested for lysis of recipient B-LCL, CD40-CLL and primary CLL cells, and against donor B-LCL and CD40 activated B cells. Data is shown at an effector to target ratio of 30:1.

Figure 4. CD8⁺ T cells reactive with the SMCY peptide FIDSYICQV develop late after NM-HSCT in UPN 22388

A Decline in total lymphocyte count in UPN 22388 in the first 28 days post-transplant. B. Recognition of HLA-A*0201 LCL from unrelated female and male donors by CD8⁺ T cell clone 13C6 isolated from PBMC obtained at day 120 after allogeneic NM-HSCT. The data is shown for an E/T of 10:1. C. Recognition by SMCY-specific clone 13C6: recipient B-LCL (◆); donor B-LCL either unpulsed (■) or pulsed (▲) with 10 μM FIDSYICQV and 3 μg/ml human β-2 microglobulin. D. SMCY-specific T cells developed late after transplant in UPN 22388. The T cell lines generated from UPN 22388 at day +63, +120, and +374 after transplant and the T cell clone 13C6 were stained with PE-conjugated anti CD3 and FITC conjugated anti CD8 monoclonal antibodies and an APC conjugated HLA-A*0201 tetramer folded with FIDSYICQV.