Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for malignant hematological diseases in adults. Due to the delayed immune reconstitution after HSCT, human cytomegalovirus (CMV) can reactivate, leading to prolonged hospitalization and increased morbidity and even mortality. Natural Killer (NK) cells have recently been described to undergo persistent reconfiguration in response to CMV-reactivation. Here we analyzed the presence and expansion of CMV-specific NK cells in patients after allogeneic HSCT.
Methods: A multicolor flow cytometry panel for monitoring the CMV-specific NK cell (NKG2C+CD57+) reconstitution and expression of activating receptors was established. Reconstitution of CMV-specific NK cells was assessed in peripheral blood samples from 67 CMV-seropositive patients. The samples were collected and analyzed between day 0 and 100 post-HSCT at intervals of 7-10 days. Monitoring of CMV-reactivation by CMV-pp65 expression and reconstitution of CMV-specific T cells (CMV-CTLs) was done routinely in our laboratory, using 7 commercially available, certified CMV-tetramers, allowing for comparison of CMV-CTL and NKG2C+CD57+ NK cells. For further immunological tests, PBMCs from CMV-seropositive healthy volunteers were isolated by density gradient centrifugation. NK cells were negatively selected by magnetic bead separation. Additional purification of NKG2C+CD57+ NK cellswas achieved by cell sorting. Selected NK cells were expanded by co-culture with irradiated allogeneic PBMCs as feeder cells and the medium was supplemented with PHA and IL-2. Expanded CD57+NKG2C+ NK cells were KIR-typed.
Our patient cohort consisted of 67 patients after allogeneic HSCT with a median age of 59 years (range: 20-75). Forty-two patients (62.7%) were transplanted for acute leukemia, 54 (80.6%) received reduced intensity conditioning (RIC) and 62 (92.5%) received anti-thymocyte-antibodies globulin (ATG). GvHD-prophylaxis was cyclosporine A (CsA) in combination with mycophenolate motefil (MMF) for 82.1% of the patients and 77.6% were transplanted from matched donors. Thirty-three (49.2%) patients reactivated CMV (median age: 59.5 years, range 28-75; median day of reactivation: 38 days post-HSCT, range: 19-54).
A significant increase in the absolute cell counts of NKG2C+CD57+ NK cells was observed after CMV reactivation, when compared to patients who did not reactivate CMV (p<0.0001). Interestingly, we observed a decreased expression of the CD8-molecule on NK cells during CMV-reactivation. CD8-expression on NK cells was previously described to be associated with a more cytotoxic phenotype of NK cells, this decrease may be a consequence of apoptosis following lytic activity. Monitoring for an additional activation marker, NKG2D, showed a significant increased expression after CMV reactivation (p=0.006), demonstrating not only the activating regulation of NK cells, but also, the co-stimulatory effects on T cell proliferation and cytokine production. Remarkably, when comparing NKG2C+CD57+ NK cells with CMV-specific T cells (Figure 1), both cell populations show similar kinetics of expansion, with an increase in the absolute cell counts during and after CMV-reactivation.
NKG2C+CD57+ preliminary expansion-studies were performed using peripheral blood samples from CMV-seropositive healthy volunteers. After two weeks in culture, an expansion of up to 3100-fold was achieved. Further studies to assess the proliferative capacity of NKG2C+CD57+ subpopulation and its functional properties post-HSCT, are ongoing. In addition, an extensive panel of cytokines and chemokines excreted by the NKG2C+CD57+cells will be studied in order to evaluate their recruitment ability of other cell-types.
Conclusion: Taken together, our results indicate that NK cells undergo a dynamic modulation and expansion of this population occurs in response to CMV-reactivation. Additionally, NKG2C+CD57+ NK cells may substitute for missing CMV-specific T cells shortly after HSCT and may play an important role in sustaining the immune reconstitution after CMV-reactivation. This study shows that NKG2C+CD57+ NK cells can be selected and expanded in vitro, holding promise for adoptive transfer in patients with recurrent CMV-reactivations.
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