Following allogeneic blood or marrow stem cell transplantation (BMSCT), donor T cells are persistently exposed to host alloantigens and develop into effector-memory (TEM) cells. CD4+ T-EM cells are thought to play a role in perpetuating chronic GVHD (cGVHD) (

K. Yamashita et al., BLOOD 103:3986, 2004
). In contrast to central memory (T-CM) and naïve CD4+ T cells, human CD4+ T-EM cells over-express the voltagegated potassium channel Kv1.3 when activated. As a result, Kv1.3 has emerged as an attractive pharmacological target for the selective suppression of T-EM cells, especially in the setting of autoimmune disease (
C. Beeton et al., Mol Pharm 67:1369, 2005
). We hypothesized that over-expression of Kv1.3 by T-EM cells obtained from allogeneic BMSCT patients with cGVHD would make them preferential targets for Kv1.3-specific potassium channel blockers. To our knowledge, no one has examined the effect of Kv1.3 blockers on T-EM cells from patients with cGVHD. In this study, we isolated T-EM cells as well as central-memory (T-CM) and naïve T cells from fresh (n = 5) and frozen (n = 4) peripheral blood (PB) of patients who had undergone allogeneic BMSCT more than 6 months earlier as well as from normal volunteers by fluorescence-activated cell sorting (FACS). All BMSCT patients had some clinical evidence of cGVHD. T-EM cells were identified by their characteristic immunophenotype: CD4+ CD45RO+/RA- CCR7- CD62L Low. The percentage of CD4+ T-EM cells in 9 patients with cGVHD was 45 ± 21% (range 19%–70%) as compared to approximately 11% in healthy controls. Sorted T-EM, T-CM and naïve CD4+ T cells were polyclonally activated ex vivo using Dynal beads coupled with anti-CD3/CD28 monoclonal antibodies. The proportion of cells secreting gamma interferon (γIFN) was quantified using EliSpot assays to assess functional activity. We first compared the kinetics of γIFN secretion by activated T-EM cells to that of T-CM and naïve T-cells. Only T-EM cells secreted γIFN within 24 hrs of activation; T-CM cells secreted γIFN ≥ 48 hrs after activation, and naïve T cells required ≥ 96 hrs. ShK peptide, a highly specific inhibitor of Kv1.3 channels derived from the sea anemone Stichodactyla helianthus, was added at the initiation of 24-hr cultures. Dose response assays confirmed that T-EM cells in both BMSCT patients and normals were preferential targets of the Kv1.3-specific peptide. On average, T-EM cells were inhibited 26%, 55%, and 69% with ShK concentrations of 1, 10 and 100 nM, respectively. With 100nM ShK, T-EM cells sorted from the peripheral blood of BMSCT patients were inhibited an average of 44 ± 21% (range 17–67%). Our data, using polyclonally activated T-EM cells, support further testing of Kv1.3 blockers against host-alloantigen-specific T-EM cells obtained from BMSCT recipients and as a potential therapeutic agent for cGVHD.

Topics:
memory, potassium channel blockers, t-lymphocytes, graft-versus-host disease, chronic, peptides, autoimmune diseases, cd28 antigens, cell separation, hematopoietic stem cell transplantation, immunophenotyping

Disclosures: No relevant conflicts of interest to declare.

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2008, The American Society of Hematology
2008
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