Page 157 - Haematologica August 2018

Unconventional CD56dim/CD16neg NK cells in HSCT
Supplementary Table S1), neither the frequency (Online Supplementary Figure S2A) nor the phenotype (data not shown) of uCD56dim NK cells were affected over time by this viral infection.
Even though the existence and the functional relevance of uCD56dim NK cells have been previously reported,15-17 a recent study claimed that the CD56dim/CD16neg phenotype is induced by cryopreservation.27 To further validate our experimental results performed on cryopreserved cells, we compared the distribution of NK cell subsets between freshly isolated and thawed PBMCs from healthy donors and transplanted patients. Our results showed that both frequencies and phenotypes of the four NK cell subsets from cryopreserved PBMCs are similar to those of freshly purified ones (Online Supplementary Figure S2B-D).
uCD56dim lymphocytes are bona fide NK cells
To confirm that CD14neg/CD3neg/CD20neg uCD56dim lym- phocytes are indeed NK cells, polychromatic flow cytom- etry data from 11 healthy donors and from five patients purified three weeks after hHSCT were labelled with a unique computational barcode, concatenated and ana- lyzed by the t-distributed Stochastic Neighbor Embedding (t-SNE) algorithm.28 We arbitrarily identified 13 different
clusters (from C1 to C13) of non-T and non-B lympho- cytes on the basis of population boundaries distinguish- able on the t-SNE density plots (Figure 2A). We then deter- mined the frequency of antigen expression in each cluster by manual gating (Online Supplementary Figure S3), and dis- played the results using a heat map. Figure 2B shows that all clusters except C13 harbor several NK cell surface markers at different degrees of expression. cCD56bright, cCD56dim and uCD56dim NK cell subsets overlap across the distinct clusters defined in the t-SNE map and show a sim- ilar distribution in healthy donors and hHSCT recipients (Figure 2C,D). cCD56bright are composed of C7, C8, C9 and, as expected, are NKp46pos and NKG2Apos while expressing low levels of Granzyme-B and Perforin. cCD56dim NK cells are present at higher frequencies in healthy donors com- pared to hHCST recipients and belong to C3, C4, C5, and C6 groups. These cells are characterized by constitutive high expressions of Granzyme-B and Perforin. uCD56dim cells are comprised of C10, C11, C12 and are NKp46neg-low, NKG2Dpos, Granzyme-Bpos and Perforinpos (Figure 2E). This high-dimensional approach of polychromatic flow cytom- etry data analysis, although applied to a small number of hHSCT patients, indicate that uCD56dim cells are bona fide NK cells.
A
DE
BC
Figure 2. Clustering of uCD56dim NK cells. (A) t-distributed Stochastic Neighbor Embedding (t-SNE) plot of lymphocytes from 11 healthy donors (HDs) and five recip- ients at three weeks after haploidentical HSCT (hHSCT). CD3pos T (green on the left plot) and CD20pos B (orange on the left plot) cells are grouped within the t-SNE map. Within the CD3neg/CD20neg gate (gray within the left plot), 13 (from C1 to C13) different clusters of lymphocytes were defined based on the population bound- aries (right plot). (B) Heatmap showing the degree of expression of CD56, CD16, CD8, NKp46, NKG2A, NKG2D, Granzyme-B (GRM-B) and Perforin on the 13 clusters of non-T and non-B lymphocytes defined in the right t-SNE plot of panel A. (C-D) t-SNE plots showing, within the 13 CD3neg/CD20neg clusters of lymphocytes presented in panel B, the clusters of cCD56bright (blue), cCD56dim (black) and uCD56dim (red) NK cell subsets from HDs and from hHSCT-patients three weeks after HSCT together (C) or separately (D). (E) Graphs showing the frequencies (median ± SEM) of cCD56bright, cCD56dim and uCD56dim from HDs and patients at three weeks after hHSCT (w3) out of the total cells in each of the 13 clusters of CD3neg/CD20neg lymphocytes.
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