The current emphasis is to describe and understand the defects in NK cell function in a group of patients with Chediak-Higashi and Hermansky-Pudlak (type 1, 2, 4 and 10) syndromes. We analyzed NK cells in PBMCs of four CHS patients with the active disease, their healthy immediate family members, as well as one CHS patient that underwent bone marrow transplantation. We found that the two CHS patients have slightly lower, but close to normal percentage of NK cells (2% and 3.3% for patient 1 and 2, vs. 4.39% and 4.06% for parents). The levels of the investigated activating and inhibitory NK cell surface receptors were normal and there was no difference between receptor levels in the two patients when compared to their family members or unrelated healthy individual, indicating that the disease is not affecting the trafficking of NK cell surface receptors. In addition, the intracellular levels of critical components of lytic granules, perforin and granzyme B, were very similar between family members. When compared to the healthy family members, however, NK cells from CHS patients had severely decreased cytotoxic potential, due to impaired degranulation ability. Surprisingly, while all NK cells had impaired degranulation, the cause underlying this defect depended on the position of LYST mutation. Mutations in the N-terminal part of the protein resulted in formation of giant lysosomes that are able to polarize to the cell-cell contact area, but are too big to be released;mutations in C-terminal part of LYST result in slightly enlarged granules that have decreased mobility, and thus impaired exocytosis. Intriguingly, the defect of release of the lytic granules was not observed in case of cytokine production and secretion. When compared to their parents, the CHS patients have similar level of up-regulation of MIP-1a and TNFa in response to cytokine stimulation. Moreover, the cytokine stimulation resulted in more robust IFNg production by NK cells of both patients (67-69% of NK cells in patients vs. 40 53% in parents). In comparison, NK cells from the CHS patient that received a bone marrow transplant appeared to be normal and the levels of the cell surface expression of NK cell activating and inhibitory receptors were also just like in healthy individuals. NK cells of the transplanted patient conjugated with target cells normally and the killing of two different target cell lines was comparable to NK cells from a healthy individual. Furthermore, NK cells of the transplanted patient readily polarized perforin and granzyme A to the cell-cell contact site. Interestingly, when compared to a healthy donor, NK cells of the transplanted patient showed increased degranulation in response to engagement of CD16 (27% vs 10% of the healthy donor) and, consequently, ADCC was increased in case of the transplanted patient. In response to cytokine stimulation, the production of MIP-1a, IFNg and TNFa by NK cells from the transplanted CHS patient was comparable to that of NK cells from a healthy donor. Thus, the bone marrow transplant fully restored NK cell functionality in this patient. We also analyzed NK cells from eight HPS-1, one HPS-2, two HPS-4, and one HPS-10 patient. NK cells from HPS type 2 and type 10 patients failed to kill target cells in natural cytotoxicity and ADCC assays, while NK cells from HPS type 1 and HPS type 4 patients had only slightly decreased capacity to kill the target cells. Conjugation of HPS NK cells to target cells did not appear to be significantly affected;F-actin accumulated at the cell-cell contact site, suggesting that the synapse formation was likely unaffected in those NK cells. However, NK cells from HPS type 2 and 10 failed to degranulate, in line with impaired cytotoxicity. Furthermore, we found that lytic granules in HPS-2 NK cells do not cluster efficiently around the MTOC, do not polarize to the IS, and appear to be slightly enlarged. HPS-10 NK cells contained large lytic granules that failed to polarize to the IS. The large lysosomes were reminiscent of the giant lysosomes observed in CHS. In several cases the large granules were positive only for perforin or granzyme A;combined with the increased size, these data suggest improper protein sorting and vesicular fusion. All HPS NK cells were able to produce cytokines (TNFa and IFNg) in response to stimulation. While HPS-1 and HPS-10 NK cells secreted normal levels cytokines, HPS-2 NK cells failed to release the cytokines following the cell stimulation. Thus, HPS-10 affects the cytolytic function of NK cells, while HPS-2 affects both lytic granule and cytokine secretion (regulated and constitutive exocytosis pathways).

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