Abstract 5115


Allogeneic stem cell transplantation (allo-SCT) is a potentially curative treatment option for patients with hematological disorders. Alloreactive donor-derived T lymphocytes exert a beneficial graft-versus-leukemia (GVL) effect through the recognition of leukemia-restricted (or preferentially expressed) antigens as Wilms tumor protein (WT1), survivin (SURV) or proteinase (PR1). Currently research in transplant immunology focuses in enhancing GVL while preventing the deleterious graft-versus-host disease (GVHD) that could be achieved by manipulating donor-derived antigen-specific T-populations. In this study we tested the presence of peripheral blood leukemia-associated antigen-specific CD8+ T-lymphocytes during post allo-SCT follow-up.


Forty-three consecutive HLA*0201 patients (homo or heterozygotous) undergoing conventional myeloablative (n=24) or non-myeloablative (n=19) allo-SCT as treatment of hematological disorders were included. Allogeneic donor was an HLA-identical sibling in 26 cases (60.5%) and unrelated in 17 cases (39.5%). Hematopoietic stem cell source included mobilized peripheral blood (n=20), bone marrow (n=18) and umbilical cord blood (n=5). As GVHD prophylaxis regimens Cyclosporine plus Methotrexate (n=20) or Cyclosporine plus Mofetil micofenolate (n=23) were employed. In addition, 22 patients received rabbit antithymocyte globulin at 6-8mg/kg. At last follow-up four patients had relapsed 9-14 months after allo-SCT. We sought for leukemia-antigen specific CD8 lymphocytes in peripheral blood samples drawn within a median of 7 months (range 2-38) when lymphocyte recovery had occurred and complete donor chimerism was achieved. We used four color multiparametric flow cytometry in a FACSCanto II acquiring at least 5 ×105 viable (Propidium Iodide low) lymphoid gated events, stained with MnAbs: CD8-FITC and CD3PE/APC MnAb. To identify leukemia-antigen specific CD8 lymphocytes we used class I HLA pentamers 0201 APC or PE conjugated (Proimmune, London, UK) against the following nonapeptides: Proteinase 1: VLQELNVTV (169-177) WT1: RMFPNAPYL (126-134) and SURV: ELTLGEFLKL (95-104). As positive staining control we used CMV pp65: NLVPMVATV (495-503) and as negative controls we used irrelevant nonapeptide and peripheral blood samples from patients lacking HLA* 0201 genotype.


Detection of donor-derived CD8+ lymphocytes against CMV pp65 occurred in 61% of recruited patients with a median percentage of 0.1% (range 0.03-13 over CD3+CD8+ events). Likewise, it was possible to detect CD8+ lymphocytes specific for PR1, WT1 and SURV in 65.2%, 47.8% y 39.1% of recruited patients respectively. Median percentage of PR1 and WT1 leukemia-antigen specific lymphocytes was 0.1% (range 0.04-1% over CD3+CD8+ events) and for WT1 0.1% (range: 0.01-0.2%). Detection of leukemia-antigen specific CD8+ lymphocytes was not significantly associated with clinical variables such as conditioning regimen (conventional or non-myeloablative), Disease status at transplant, donor type (sibling or unrelated), ATG use or HLA-disparity degree. The presence of WT1 specific CD8+ lymphocytes was significantly more frequent in patients undergoing allo-SCT for lymphoid hematological malignancy (p=0.04). By contrast, the presence of circulating anti-PR1 specific CD8+ lymphocytes was not more frequently found in patients undergoing allo-SCT for myeloid malignancies. Of note, none of the four patients who eventually relapsed harbored circulating leukemia-specific CD8+ lymphocytes.


Multiparametric flow cytometry is a useful tool to detect and quantify rare donor-derived CD8+ lymphocytes specific for leukemia-associated antigens as PR1, WT1 or SURV. The presence of these populations in peripheral blood is not associated to conventional clinical variables and in our series anti-WT1 CD8+ lymphocytes were more frequently detected in patients receiving allo-SCT for lymphoid malingnacies. By contrast, larger series are needed to assess if the lack of these leukemia-associated antigen-specific CD8 lymphocytes in peripheral blood could identify patients in a higher risk of relapse.

Financial support

This study was supported by a grant of Conserjeria de Salud, Junta de Andalucia 2006/0355. J. Serrano López is a post-doc fellow from Fundación Española de Hematología y Hemoterapia


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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