Human NK cells modulate NCAM protein expression and degree of polysialylation with activation state
Flow cytometric analyses showed that human PBMCs expressed the NCAM protein scaffold and its polysialic acid modifications, which were sensitive to the polySia-specific neuraminidase Endo N (). Upon activation with IL-2, the cell-surface levels of both the underlying protein and the attached glycan increased (; n = 10). To address whether the observed increase in polySia expression reflected differential proliferation of CD56dim and CD56bright NK cell populations during the assay, sorted cells with these phenotypic characteristics were analyzed in parallel; similar responses were observed (data not shown). These data suggest that polySia levels are regulated by NK cell activation.
NCAM/CD56+ human NK cells modulate polySia and NCAM expression with activation
As the DP of polySia on human primary cells has not been explored, purified NK cells were cultured with or without IL-2 for 48 hours. Samples from five individual donors were combined for analysis. Intact polySia chains on glycan cores containing lactosamine were released by Endo-β-galactosidase treatment. This method may have neglected to free polySia that was attached to NCAM through alternate core structures, however the relative amounts of total sialic acid released from resting and activated NK cells was in accord with our flow cytometry () and immunoblotting data (not shown). The liberated polySia was analyzed by high-performance liquid chromatography (HPLC) as previously described to determine chain length (2
). Total sialic acid content [as N
-acetyl neuraminic acid (NANA)] of HPLC fractions was monitored to track the relative abundance of each DP population. In both resting and activated NK cells, a large fraction of total NANA was associated with small non-polySia glycans (DP 1–10), most likely reflecting the capping groups of typical N- and O-linked glycans. Plotting percent of total NANA versus DP showed that activation of NK cells increased the abundance of NANA associated with polySia (). Additionally, chain length, which was extremely heterogeneous on resting cells, was compressed into the mid-range (DP 11–140) upon activation.
Human NK cell activation results in an increased production of short to medium-length polySia chains
ST8Sia IV catalyzes polySia expression by populations of mouse bone marrow cells during myeloid differentiation
To analyze the potential immunological properties of polySia, we used a mouse model. First, we characterized the expression patterns of polySia on immune subsets in wild-type animals. In contrast to their human counterparts, polySia was not detectable on mouse NK cells (). This finding was consistent with RT-PCR analyses on sorted NK cells that revealed an absence of NCAM and ST8Sia IV (data not shown). Interestingly, robust polySia expression was detected on wild-type mouse bone marrow subsets (). This expression was conserved in ST8Sia II-/- but absent in ST8Sia IV-/- mice, indicating that the latter enzyme was responsible for polySia on these cells.
ST8Sia IV produces polySia on subsets of mouse bone marrow and peripheral myeloid cells
Bone marrow polySia expression correlated with receptor tyrosine kinase cKit expression, suggesting that the polySia+ cells were hematopoietic progenitors. We defined four subsets according to their relative levels of polySia (PSA) and cKit (Kit) (PSAneg/Kithi, PSAlo/Kithi, PSAhi/Kithi, PSAlo/Kitlo; ). The populations comprised 12%, 6%, 12% and 42%, respectively, of total cKit+ bone marrow cells. Extensive phenotypic analyses by flow cytometry suggested that these subsets comprised cells that were differentiating along a myeloid pathway (). The PSAneg/Kithi population included hematopoietic stem cells (HSCs) (defined as Lin-, cKit+, Sca-1+) and the majority appeared to be progenitors as they were Lin- and expressed CD34. The second population (PSAlo/Kithi) comprised more committed progenitors with near uniform expression of CD34, and to a lesser extent CD11b and Gr-1. As differentiation proceeded, evidenced by a reduction in CD34 expression and an increase in CD11b and Gr-1 expression, PSA levels dramatically increased in parallel (PSAhi/Kithi). The fully differentiated progeny, (CD34+/-, CD11b+, Gr-1hi) had polySia levels that were comparable to the progenitors (PSAlo/Kitlo). Consistent with the latter population containing mature myeloid cells, we found low levels of polySia expression on peripheral wild-type, but not ST8Sia IV-/-, Gr-1+ splenocytes (). Thus, myeloid differentiation is characterized by a wave of high levels of polySia expression. In contrast to the mouse, human fetal bone marrow did not contain these polySia+ myeloid populations, and human peripheral myeloid cells did not express polySia (data not shown). The polySia+ subset in human fetal bone marrow consisted of NK cells, as determined by the phenotype: CD56+, CD7+, CD33- and CD34- (data not shown). It is worth noting that the observed differences between adult mouse bone marrow and fetal human bone marrow may be attributable to variations in polySia expression during development.
In vitro progenitor studies confirm the phenotypic analyses of the polySia expressing subsets
To confirm the phenotypic analyses, the bone marrow populations defined by polySia and cKit were sorted by flow cytometry and tested in vitro and in vivo for their ability to give rise to various immune lineages. For these experiments, the PSAneg/Kithi subset served as a positive control, as hematopoietic stem cells were contained in this population (data not shown). In colony-forming assays testing erythroid and myeloid potential, both the positive control and the progenitor subset, PSAlo/Kithi, gave rise to these lineages, forming both erythroid blasts and colonies, and myeloid colonies and clusters (). Of the more differentiated populations, the immature myeloid cells, PSAhi/Kithi, showed an intermediate ability to form myeloid clusters, and did not produce erythroid populations. The fully differentiated PSAlo/Kitlo cells did not produce colonies in either assay.
The PSAneg/Kithi and PSAlo/Kithi subsets contain erythroid and myeloid progenitors
To test lymphoid progenitor potential, sorted bone marrow subsets were co-cultured with OP9 cells transfected with a GFP vector control, or with GFP and the Notch ligand, Delta-like 1 (DL1). In this assay, multipotent progenitors develop into B cells when cultured on OP9-GFP feeders, while DL1 induces T-cell development. NK cells develop at lower efficiencies under both conditions. In accord with the phenotypic data and colony-forming assays, the positive control, PSAneg/Kithi, and the progenitor population, PSAlo/Kithi, produced all the lymphoid lineages, the latter with a 10-fold reduction in efficiency as compared to the former (). The immature myeloid subset, PSAhi/Kithi, did not produce NK or T cells, and generated almost 1000-fold fewer B cells than the positive control population. The mature myeloid population, PSAlo/Kitlo, did not proliferate.
The PSAneg/Kithi and PSAlo/Kithi subsets contain lymphoid progenitors
In vivo progenitor studies confirm the phenotypic and in vitro analyses of the polySia expressing subsets
For in vivo experiments, GFP+ congenic wild-type mice were used as donors so that engrafted lineages could be identified using this fluorescent reporter. Flow-sorted bone marrow subsets (~28,000 cells/mouse) were injected with a survival dose (1 × 106 cells/mouse) of wild-type bone marrow into irradiated wild-type recipient animals. Three weeks later mice were sacrificed and organs analyzed for GFP+ populations (). Both the positive control, PSAneg/Kithi, and the progenitor population, PSAlo/Kithi, gave rise to erythroid (TER119+), myeloid (Gr-1+ and CD14+) and lymphoid (DX5+ and B220+) cells in the bone marrow and spleen. No statistically significant differences were noted in the numbers of cells recovered from these two donor populations. In contrast, the numbers of GFP+ cells isolated from the immature myeloid subset, PSAhi/Kithi, and the mature myeloid population, PSAlo/Kitlo, were reduced by an average of ~ 40-fold and ~ 500-fold, respectively, as compared to the progenitor subsets.
Next, we asked whether development of the four bone marrow subsets was temporally linked. Mice were treated with one bolus of 5-fluorouracil to deplete cycling cells, and the disappearance and reappearance of the polySia/cKit-defined populations was followed over 12 days. As expected, the depletion and recovery of the progenitors preceded by one to two days that of the mature subsets ().
Development of the polySia (PSA)/cKit-defined subsets is temporally linked
Finally, we stimulated myeloid development and characterized the response of the bone marrow subsets. Briefly, wild-type mice were injected every 24 hours with granulocyte-colony stimulating factor (G-CSF) for five days to induce expansion of the myeloid compartment, and bone marrow was analyzed by flow cytometry. In accord with the preceding phenotypic, in vitro and in vivo data, both the progenitor population, PSAlo/Kithi and the immature and mature myeloid subsets (PSAhi/Kithi and PSAlo/Kitlo) significantly expanded in response to G-CSF treatment as compared to vehicle-treated control mice (p < 0.01; ). Collectively, the data in and – reveal that polySia expression is expressed and modulated during myeloid development in the mouse.
G-CSF increases the production of the PSAlo/Kithi, PSAhi/Kithi and PSAlo/Kitlo subsets
Given the expression of polySia on hematopoietic progenitors and myeloid cells, we asked whether there were obvious changes in the distribution of immune subsets in ST8Sia IV-/- as compared to wild-type mice. Hematological analyses revealed minimal differences—a statistically significant, slight increase in the percentage of lymphocytes in peripheral blood and a corresponding though not significant decrease in the percentage of circulating monocytes and neutrophils (data not shown). No changes in the numbers or phenotype of peripheral myeloid lineages were noted (data not shown).
NCAM is the scaffold for myeloid expression of polySia
As this study was the first description of polySia on myeloid cells, the underlying protein scaffold was unknown. Candidate proteins that are expressed by other cell types that carry polySia modifications include NCAM and CD36. To identify the scaffold on the myeloid subsets that were the subject of this investigation, we immunoprecipitated wild-type and ST8Sia IV-/- bone marrow lysates with an anti-polySia antibody, treated the precipitatate with Endo N to remove polySia, and separated the deglycosylated proteins by SDS-PAGE. Following electrophoresis, two silver stained bands of approximately 120 and 140 kDa were observed in the wild-type, but not the ST8Sia IV-/- samples (). The bands were excised and analyzed by electrospray mass spectrometry (LTQ linear ion trap), which identified both bands, on the basis of two peptides each, as NCAM (data not shown). In accord with this finding, both flow cytometry and immunoblotting confirmed the presence of NCAM on the relevant cells in mouse bone marrow ().
NCAM on the surface of mouse bone marrow cells is the scaffold for polySia
ST8Sia IV-/- mice exhibit exaggerated contact hypersensitivity and an inability to control growth of engrafted tumors
Next we tested immune responses in ST8Sia IV-/- mice. First, we used a contact hypersensitivity (CHS) assay. Wild-type and ST8Sia IV-/- mice were sensitized with the hapten dinitrofluorobenzene (DNFB); a week later, they were challenged with an application of DNFB to one ear, and vehicle alone to the other ear. Wild-type mice responded as expected, with peak swelling observed around 24 hours (23
). Interestingly, the ST8Sia IV-/- response equaled or exceeded the wild-type response at 24 hours, and inflammation continued to increase through 48 and 72 hours. shows the results of a representative experiment in which ST8Sia IV-/- ear thickness was statistically increased at all timepoints as compared to the response of wild-type animals.
The contact hypersensitivity response is excessive in ST8Sia IV−/− mice
We also assessed the CHS response at 72 hours on a histological level, and noted significantly more inflammation and edema after DNFB treatment of ST8Sia IV-/- mice as compared to controls, whose lesions were much less severe (). Injury to the epithelium of ST8Sia IV-/- ears was also more pronounced, with diffuse epidermal hyperplasia, intercellular edema and ulceration. The ST8Sia IV-/- vehicle-treated ears also demonstrated minimal to mild edema whereas the wild-type controls had no significant lesions.
We used immunosensitive and immunoresistant cell lines to test the response of ST8Sia IV-/- mice to a tumor challenge. RMA-S cells, which have reduced MHC class I expression, are sensitive to NK killing, while the parental RMA cells form tumors in wild-type animals (24
). In initial experiments, RMA-S cells formed tumor masses in NK-cell-depleted wild-type animals (25
), but not wild-type or ST8Sia IV-/- mice. This finding suggested that the NK-cell compartment of ST8Sia IV-/- mice was intact (data not shown). In contrast, injection of RMA cells into wild-type, ST8Sia IV-/- or the immunodeficient TCRβ-/- mice led to uncontrolled tumor growth in all cohorts, requiring euthanasia of the animals when their tumors exceeded acceptable size limitations (). Importantly, tumor growth in ST8Sia IV-/- mice was significantly faster than in wild-type mice, and was comparable to the rate observed in TCRβ-/- mice (p < 0.02).
RMA tumors grow faster in ST8Sia IV−/− mice than in wild-type animals