Xuzhang Lu

Mycophenolic Acid Inhibits Natural Killer Cell Proliferation and Cytotoxic Function: A Possible Disadvantage of Including Mycophenolate Mofetil in the Graft-Versus-Host Disease Prophylaxis Regimen

To determine how immunosuppressant agents used for graft-versus-host disease (GVHD) prophylaxis affect natural killer (NK) cells, we examined the effects of cyclosporine (CSP), tacrolimus (TAC), mycophenolic acid (MPA, an active form of mycophenolate mofetil), and methotrexate (MTX) on the proliferation and cytotoxicity of NK cells. The proliferation of NK cells from healthy individuals in the presence of interleukin (IL)-2 and IL-15 was suppressed to 51% ± 16% of that of the controls with CSP, to 31% ± 19% with TAC, to 14% ± 6% with MPA, and to 87% ± 18% with MTX. Both CSP and TAC increased the proportion of CD16(-)CD56(bright) cells, a NK cell subset capable of secreting high amount of cytokines, and also enhanced NKp30 expression, whereas MPA markedly decreased the proportion of CD16(-)CD56(bright) cells and reduced the expression of all activating NK cell receptors, including NKG2D, NKp30, NKp44, and NKp46. MPA also reduced the cytotoxicity against K562 cells from 61% ± 15% to 17% ± 7% and that against Daudi cells from 44% ± 4% to 4% ± 4%, whereas the other 3 drugs did not diminish these cytotoxicities. The inhibition of NK cell proliferation and cytotoxicity against leukemic cell lines by MPA was partially abolished by the inclusion of guanosine in the culture. Similar to the effect of MPA on T cells, MPA inhibited the down-regulation of p27 on NK cells induced by the incubation of NK cells in the presence of IL-2. These results suggest that MPA is a potent inhibitor of NK cells, and that its inclusion in the GVHD prophylaxis regimen might diminish the graft-versus-leukemia effect of NK cells.

Anti-Moesin Antibodies in the Serum of Patients with Aplastic Anemia Stimulate Peripheral Blood Mononuclear Cells to Secrete TNF-α and IFN-γ

Moesin is an intracellular protein that links the cell membrane and cytoskeleton, while also mediating the formation of microtubules and cell adhesion sites as well as ruffling of the cell membrane. To determine the roles of anti-moesin Abs derived from the serum of patients with aplastic anemia (AA) in the pathophysiology of bone marrow failure, we studied the expression of moesin on various blood cells and the effects of anti-moesin Abs on the moesin-expressing cells. The proteins recognized by anti-moesin mAbs were detectable on the surface of T cells, NK cells, and monocytes from healthy individuals as well as on THP-1 cells. The peptide mass fingerprinting of the THP-1 cell surface protein and the knock-down experiments using short hairpin RNA proved that the protein is moesin itself. Both the anti-moesin mAbs and the anti-moesin polyclonal Abs purified from the AA patients’ sera stimulated THP-1 cells and the PBMCs of healthy individuals and AA patients to secrete 60–80% as much TNF-α as did LPS 100 ng/ml. Although the polyclonal Abs induced IFN-γ secretion from the PBMCs of healthy individuals only when the PBMCs were prestimulated by anti-CD3 mAbs, the anti-moesin Abs were capable of inducing IFN-γ secretion from the PBMCs of AA patients by themselves. Anti-moesin Abs may therefore indirectly contribute to the suppression of hematopoiesis in AA patients by inducing myelosuppressive cytokines from immunocompetent cells.

Hydroxyurea upregulates NKG2D ligand expression in myeloid leukemia cells synergistically with valproic acid and potentially enhances susceptibility of leukemic cells to natural killer cell-mediated cytolysis.

(Cancer Sci 2010; 101: 609–615)

CD16+ CD56− NK cells in the peripheral blood of cord blood transplant recipients: a unique subset of NK cells possibly associated with graft-versus-leukemia effect

A marked increase in CD16+ CD56− NK cells in the peripheral blood (PB) was observed in a cord blood transplant (CBT) recipient with refractory acute myeloid leukaemia (AML) in association with attaining molecular remission. CD16+ CD56− NK cells isolated from the patient became CD16+CD56+NKG2D+ when they were cultured in the presence of IL‐2. Although cultured CD16+CD56− NK cells retained the killer‐cell immunoglobulin receptor (KIR)‐ligand (KIR‐L) specificity and the patient’s leukemic cells expressed corresponding KIR ligands, they killed patient’s leukemic cells expressing ULBP2. The cytotoxicity by cultured CD16+CD56− NK cells was abrogated by anti‐ULBP2 antibodies. When leukemic cells obtained at relapse after CBT were examined, both the ULBP2 expression and susceptibility to the cultured NK cells decreased in comparison to leukemic cells obtained before CBT. An increase in the CD16+CD56− NK cell count (0.5 × 109/L or more) in PB was observed in seven of 11 (64%) CBT recipients but in none of 13 bone marrow (BM) and eight peripheral blood stem cell (PBSC) transplant recipients examined during the similar period after transplantation. These findings suggest an increase in CD16+CD56− NK cells to be a phenomenon unique to CBT recipients and that mature NK cells derived from this NK cell subset may contribute to the killing of leukemic cells expressing NKG2D ligands in vivo.

Autoantibodies specific to hnRNP K: a new diagnostic marker for immune pathophysiology in aplastic anemia

To identify a new diagnostic marker for the immune pathophysiology of aplastic anemia (AA), we screened sera of immune-mediated AA patients for the presence of antibodies (Abs) specific to proteins derived from a leukemia cell line UT-7 using two-dimensional electrophoresis followed by immunoblotting. The target proteins were identified by peptide mass fingerprinting. Heterogeneous nuclear ribonucleoprotein (hnRNP) K was identified as a novel autoantigen. An enzyme-linked immunosorbent assay revealed high titers of anti-hnRNP K Abs in 85 (31%) of 273 patients with AA. Sixty-four patients received antithymocyte globulin and cyclosporine after undergoing screening for anti-hnRNP K Ab, anti-DRS-1 Ab, anti-moesin Ab, and paroxysmal nocturnal hemoglobinuria (PNH)-type cells. Twenty (87%) of 23 patients with the presence of anti-hnRNP K Abs responded to the immunosuppressive therapy (IST), while 19 (46%) of 41 patients without the presence of anti-hnRNP K Abs responded. A multivariate analysis showed only PNH-type cells and anti-hnRNP K Abs to be significant factors for the prediction of a good response to IST. The detection of anti-hnRNP K Abs as well as PNH-type cells may therefore be useful for diagnosing the immune pathophysiology of AA.

Expansion of donor-derived hematopoietic stem cells with PIGA mutation associated with late graft failure after allogeneic stem cell transplantation

A small population of CD55(-)CD59(-) blood cells was detected in a patient who developed donor-type late graft failure after allogeneic stem cell transplantation (SCT) for treatment of aplastic anemia (AA). Chimerism and PIGA gene analyses showed the paroxysmal nocturnal hemoglobinuria (PNH)-type granulocytes to be of a donor-derived stem cell with a thymine insertion in PIGA exon 2. A sensitive mutation-specific polymerase chain reaction (PCR)-based analysis detected the mutation exclusively in DNA derived from the donor bone marrow (BM) cells. The patient responded to immunosuppressive therapy and achieved transfusion independence. The small population of PNH-type cells was undetectable in any of the 50 SCT recipients showing stable engraftment. The de novo development of donor cell-derived AA with a small population of PNH-type cells in this patient supports the concept that glycosyl phosphatidylinositol-anchored protein-deficient stem cells have a survival advantage in the setting of immune-mediated BM injury.