Up-regulation of activating and inhibitory NKG2 receptors in allogeneic and autologous hematopoietic stem cell grafts

Background Hematopoietic Stem Cell Transplantation (HSCT) is known to induce the inhibitory immune receptor NKG2A on NK cells of donor origin. This occurs in allogeneic recipients, in both the haploidentical and HLA-matched settings. Methods To gain further insight, not only NKG2A, but also the activating receptors NKG2C and NKG2D were assessed by flow cytometry. Immunophenotyping was carried out not only on CD56+ but also on CD8+ lymphocytes from leukemia and lymphoma patients, receiving both HLA-matched (n = 7) and autologous (n = 5) HSCT grafts. Moreover, cognate NKG2 ligands (HLA-E, MICA, ULBP-1, ULBP-2 and ULBP-3) were assessed by immunohistochemistry in diagnostic biopsies from three autotransplanted patients, and at relapse in one case. Results All the NKG2 receptors were simultaneously up-regulated in all the allotransplanted patients on CD8+ and/or CD56+ cells between 30 and 90 days post-transplant, coinciding with, or following, allogeneic engraftment. Up-regulation was of lesser entity and restricted to CD8+ cells in the autotransplantation setting. The phenotypic expression ratio between activating and inhibitory NKG2 receptors was remarkably similar in all the patients, except two outliers (a long survivor and a short survivor) who surprisingly displayed a similar NKG2 activation immunophenotype. Tumor expression of 2 to 3 out of the 5 tested NKG2 ligands was observed in 3/3 diagnostic biopsies, and 3 ligands were up-regulated post-transplant in a patient. Conclusions Altogether, these results are consistent with a dual (activation-inhibition) NK cell re-education mode, an innate-like T cell re-tuning, and a ligand:receptor interplay between the tumor and the immune system following HSCT including, most interestingly, the up-regulation of several activating NKG2 ligands. Turning the immune receptor balance toward activation on both T and NK cells of donor origin may complement ex vivo NK cell expansion/activation strategies in unmanipulated patients. Electronic supplementary material The online version of this article (doi:10.1186/s13046-015-0213-y) contains supplementary material, which is available to authorized users.

and Y, as described [1], using a LightCycler 2.0 (Roche). The glyceraldehyde phosphate dehydrogenase (GAPDH) gene was included as a non-polymorphic gene. Reference amplification curves were obtained by running in parallel QRT-PCR using as templates mixtures containing known relative amounts of donor and recipient DNAs. Negative controls (no template DNA) were invariably included, and all determinations were performed in triplicate. The intensities of all the donor-specific and recipient-specific amplimers were averaged, and % chimerism was expressed as the relative amount of donor DNA in sample DNA.

Simultaneous up-regulation of NKG2A, NKG2C and NKG2D
To determine whether or not NKG2 receptors are simultaneously up--regulated, flow cytometry data were elaborated as follows. First, we multiplied the percent positive by the mfi values of NKG2A, NKG2C and NKG2D flow cytometry determinations. The resulting products were calculated separately for CD8 and CD56 cells at T0 (before transplant), T1 (30 days after transplant) and T2 (90 days after transplant). By this means, a product slant value was assigned to each of the white, grey and black dots visible in Figs. 2 and 4. Next, the Pearson's correlation coefficients were calculated (by Microsoft Excel) among series of three product slant values (NKG2A vs NKG2C; NKG2A vs NKG2D and NKG2C vs NKG2D). The results are reported in Table S2A. From this Table, it may be appreciated that in most cases correlations are strong (>0.7), but weak or even inverse correlations may occasionally be seen. It may be concluded that up--regulation of NKG2A, NKG2C and NKG2D is in most cases simultaneous. Table S2B reports the same analysis for autotransplanted patients. The results are similar although correlations are weaker. Table  S3 shows the mfi values, Mean values and Standard Deviations of CD56 stain at the T0 (before transplant) and T90 (90 days post--transplant) time points for all the patients. It clearly appears that CD56 stain is brighter at T90 than T0, although a two--tailed Student t test (paired) shows that differences in CD56 expression is significant only in allotransplanted patients.

Increase in CD56 expression post transplant
CD56 up--regulation, in combination with the phenotypically detectable increases in NKG2 and decrease in KIR2DL expression (see text in the paper), is consistent with an 'immature' NK cell phenotype during engraftment, and with a deeper NK cell retuning in allotransplanted as compared to autotransplanted patients.

NKG2 up-regulation and clinical data
To empirically estimate the activation/inhibition balance, the sum of the slant product values Only pt. 155 (multiple myeloma) and pt. 187 (AML) clustered apart due to both high R(act/inh) values, and their longest and shortest survival times within the allotransplantation and autotransplantation groups, respectively ( Fig. S1A and S1B). Thus, prevalence of activating receptor expression was observed in these two clinical outliers.   Table S3 CD56 mean fluorescence intensities of PBMCs from allotransplanted and autotransplanted patients at T0 (before transplant) and T90 (day 90 after transplant).

Figure S1
NKG2 up-regulation and clinical data R(act/inh) vs Overall Survival (OS) for allotransplanted and autotransplanted patients are separately displayed in panels A and B, respectively. For R(act/inh) calculation see Supplemental Results.
Each dot represents a single patient, identified by number. Two outliers are indicated.