Nancy F. Hensel

Nonmyeloablative allogeneic stem cell transplantation has recently been explored as a safer alternative to conventional high-dose transplant regimens. Although a high incidence of mixed chimerism after nonmyeloablative procedures has been reported, the exact kinetics of engrafting donor cells in specific cellular lineages has yet to be defined. We investigated lineage-specific chimerism in 15 patients receiving an allogeneic peripheral blood stem cell (PBSC) transplant from an HLA-identical (n = 14) or a 5/6 antigen-matched sibling donor after a preparative regimen of cyclophosphamide and fludarabine. Donor chimerism was assessed weekly in T lymphocytes and myeloid cells by polymerase chain reaction (PCR) of minisatellite regions. Eight patients survived between 121 to 409 days after transplant. Ten of 14 patients surviving more than 30 days (71.4%) had delayed disease regression consistent with a graft-versus-malignancy (GVM) effect. One patient rejected the transplant with subsequent recovery of autologous hematopoiesis. Hematological recovery was rapid (median, 11 days to >/=500 neutrophils/microL) and was initially predominantly recipient in origin. Donor myeloid chimerism gradually supplanted recipient hematopoiesis and became fully donor in all survivors by 200 days after transplantation. In contrast, T-cell engraftment was more rapid, with full chimerism in 7 patients by day 30 and in 6 further patients by day 200 after cyclosporine withdrawal and donor lymphocyte infusion. Full donor T-cell engraftment preceded donor myeloid engraftment, acute graft-versus-host disease, and disease regression, consistent with a requirement for 100% donor T-cell chimerism for full expression of the alloresponse. These results emphasize the importance of lineage-specific chimerism analysis to successfully manipulate engraftment after nonmyeloablative allogeneic PBSC transplantation.

Regulatory T cells (T(reg)s) that constitutively express FOXP3 are instrumental to the maintenance of tolerance and may suppress graft-versus-host disease (GVHD) in humans. To determine whether regulatory T cells in allogeneic stem cell transplants (SCTs) ameliorate GVHD after transplantation, we quantitated the coexpression of FOXP3 on CD4(+) T cells in 32 donor SCTs infused into HLA-matched siblings and examined GVHD incidence in respective recipients. High CD4(+)FOXP3(+) T-cell count in the donor was associated with a reduced risk of GVHD. We monitored T(reg)s during immune reconstitution in 21 patients with leukemia undergoing a T-cell-depleted allogeneic SCT. Early after SCT, there was a significant expansion in the CD4(+)FOXP3(+) T-cell compartment. A low CD4(+)FOXP3(+) T-cell count early after SCT (day 30) was associated with an increased risk of GVHD, and the ratio of CD4(+)FOXP3(+) T cells to CD4(+)CD25(+)FOXP3(-) T cells was significantly reduced in patients with GVHD, suggesting diminished control of effector T cells. Our findings suggest that graft T(reg) content may predict for risk of GVHD after SCT. Determining the T(reg) levels in the donor and manipulating T(reg)s early after transplantation may provide a new approach to controlling GVHD.

Proteinase 3 is present in high concentration in the primary granules of acute and chronic myeloid leukemia blasts, and may represent a potential T-cell target antigen. We screened proteinase 3 against the binding motif of HLA-A2.1. Based on its high predicted binding, a 9-mer peptide, "PR-1," was synthesized and tested for binding to HLA-A2.1 using the T2 cell line. PR-1 at 100 micrograms/mL significantly increased expression of HLA-A2.1, with median channel of fluorescence increasing from 22 to 294. Binding half-life was determined to be 1,460 minutes by I125-labeled beta 2-microglobulin incorporation. HLA-A2.1+ peripheral blood mononuclear cells from a normal donor were used to generate a T-cell line specific for PR-1. The line demonstrated 85% PR-1-specific lysis at an E:T ratio of 50:1, compared with 20% lysis without PR-1, using T2 cells as targets. It also showed 79% specific lysis to fresh chronic myelogenous leukemia blasts, 54% to fresh acute myelogenous leukemia blasts, and only background lysis (< 20%) to HLA-A2.1+ normal allogeneic marrow cells. The amount of lysis of HLA-A2.1+ myeloid cells was proportional to cytoplasmic proteinase 3 expression. Thus, HLA-A2.1-restricted cytotoxic T cells, raised against a peptide contained in proteinase 3, preferentially lysed fresh human leukemic cells.

We previously showed that a peptide (PR1) derived from the primary granule enzyme proteinase 3 induced peptide specific cytotoxic T lymphocytes (CTL) in a normal HLA-A2.1+ individual. These CTL showed HLA-restricted cytotoxicity to myeloid leukemias (which overexpress proteinase 3). To further investigate their antileukemic potential, we studied the ability of PR1-specific CTL, derived from two HLA-A2.1+ normal individuals, to inhibit colony-forming unit granulocyte-macrophage (CFU-GM) from normal and leukemic individuals. CTL from 20 day PR1 peptide-pulsed lymphocyte cultures showed 89% to 98% HLA-A2.1-restricted colony inhibition of chronic myeloid leukemia targets. Colony formation in normal HLA-A2.1+ bone marrow or HLA-A2.1- CML cells was not inhibited. Sequencing of the exon encoding PR1 showed that colony inhibition was not caused by polymorphic differences in proteinase 3 between effectors and targets. Analysis by flow cytometry showed that proteinase 3 was overexpressed in the leukemia targets compared with normal marrow targets (median channel fluorescence 1,399 v 298, P = .009). These results show that PR1-specific allogeneic T cells preferentially inhibit leukemic CFU-GM based on overexpression of proteinase 3, and that proteinase 3-specific CTL could be used for leukemia-specific adoptive immunotherapy.

Antigens implicated in the graft-versus-leukemia (GVL) effect in chronic myeloid leukemia (CML) include WT1, PR1, and BCR-ABL. To detect very low frequencies of these antigen-specific CD8+ T cells, we used quantitative polymerase chain reaction (qPCR) to measure interferon-gamma (IFN-gamma) mRNA production by peptide-pulsed CD8+ T cells from HLA-A*0201+ healthy volunteers and from patients with CML before and after allogeneic stem cell transplantation (SCT). Parallel assays using cytomegalovirus (CMV) pp65 tetramers demonstrated the IFN-gamma copy number to be linearly related to the frequency of tetramer-binding T cells, sensitive to frequencies of 1 responding CD8+ T cell/100 000 CD8+ T cells. Responses to WT1 and PR1 but not BCR-ABL were detected in 10 of 18 healthy donors. Responses to WT1, PR1, or BCR-ABL were observed in 9 of 14 patients with CML before SCT and 5 of 6 after SCT, often to multiple epitopes. Responses were higher in patients with CML compared with healthy donors and highest after SCT. These antigen-specific CD8+ T cells comprised central memory (CD45RO+CD27+CD57-) and effector memory (CD45RO-CD27-CD57+) T cells. In conclusion, leukemia-reactive CD8+ T cells derive from memory T cells and occur at low frequencies in healthy individuals and at higher frequencies in patients with CML. The increased response in patients after SCT suggests a quantitative explanation for the greater effect of allogeneic SCT.