CD19-NKCEs efficiently promote NK-cell antileukemia activity in a transplantation setting. A, Evaluation of NKp46, NKp30, CD16, perforin, and granzyme B expression in NK cells from transplanted patients (Post1–3M; n = 12) and healthy donors (HD; n = 5–11). B, Representative experiment of cytototoxicity (7AAD/AnnV staining). MHH-CALL-4 or ALL#06 primary leukemia target cells were cultured either alone (only target) or with resting NK cells from a transplanted patient (3 months after haplo-HSCT) and the indicated NKCEs (100 μg/mL). Numbers indicate the percentage of cells in each quadrant. Percent of specific lysis (7AAD/AnnV staining; C) and CD107a degranulation of resting NK cells (D) from transplanted patients against MHH-CALL-4 (left) or primary leukemia blasts (ALL#04 and ALL#06; right) in the presence of the indicated NKCE at 100 μg/mL. Pooled data obtained with primary leukemia blasts are shown. Results from 5–8 independent experiments are reported. The incubation time for all the tests was 4 hours. Bar show mean ± SEM. Statistical significance: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Mann–Whitney test was used to calculate statistical differences.

Effect of NKCEs targeting CD19 or CD20 on BCP-ALL cell lines. A, Two pediatric BCP-ALL cell lines were characterized for the surface expression of CD19 and CD20 via flow cytometry using specific mAbs followed by PE-conjugated anti-IgG1 secondary reagent. Numbers represent the percentage of positive cells. B, Comparison of cytotoxicity (51Cr-release assay) of resting NK cells from healthy donors against MHH-CALL-4 (top row, n = 2–6) or NALM-16 (bottom row, n = 3) cells in the presence of NKCEs at the indicated concentrations (see legend for color symbols in D). E:T ratios were 10:1 and 5:1 for MHH-CALL-4 cells and NALM-16 cells, respectively. Percent of specific lysis, via 7AAD/AnnV staining, of MHH-CALL-4 cells cocultured with resting NK cells from healthy donors (n = 4–9) in the presence of CD19-NKCEs or IC-NKCEs (C), and CD20-NKCEs, IC-NKCEs, or rituximab (D) at 10−2 μg/mL and 100 μg/mL. Results from 6–10 independent experiments are reported. In both cytotoxicity assays, 4-hour coculture was performed. Bars show mean ± SEM. Statistical significance: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Two-way ANOVA followed by Tukey test was used to calculate statistical differences among the indicated NKCEs within each concentration. Mann–Whitney test was used to compare each NKCE at the two indicated concentrations.

NKCEs enhance NK-cell activity against MHH-CALL-4 cell line. CD107a expression and IFNγ production by resting NK cells from healthy donors (n = 3–6) cocultured for 6 hours with MHH-CALL-4 cells in the absence or presence of CD19-NKCEs, CD20-NKCEs, or the control molecules (IC-NKp46-NKCE, IC-NKp30-NKCE) at 10−2 μg/mL and 100 μg/mL. A, Flow cytometry of a representative experiment is shown. Numbers indicate the percentage of cells in each quadrant. CD107a expression (B) and IFNγ production (C) were performed in 3–6 independent experiments. Bars show mean ± SEM. Statistical significance: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Two-way ANOVA followed by Tukey test was used to calculate statistical differences among the indicated NKCEs within each concentration. Mann–Whitney test was used to compare each NKCE at the two indicated concentrations.

Effect of NKCEs targeting CD19 or CD20 on primary BCP-ALL cells. A, Phenotypic characterization via flow cytometry of four BCP-ALL primary leukemia samples, using CD45-APC-Vio770, CD19-PE-Cy7, and CD20-V450. B, 7AAD/AnnV staining of cells from sample ALL#06 cultured either alone (only ALL#06) or with resting NK cells from a representative healthy donor and the indicated NKCEs (100 μg/mL). C, CD107a expression of NK cells cultured either alone or with cells from ALL#06 in the presence of IC-NKp46-NKCE, CD19-NKp46-NKCE, or CD20-NKp46-NKCE (100 μg/mL). A representative experiment is shown. Numbers indicate the percentage of cells in each quadrant. D, Percent of specific lysis of CD19+ leukemia blasts (left) and CD107a degranulation (right) of resting NK cells from healthy donors (n = 3–6) upon coculture with primary leukemia blasts and NKCEs at 100 μg/mL as indicated. Data obtained with target cells containing leukemia blasts <85% (ALL#02 and ALL#07) or >85% (ALL#04 and ALL#06) were pooled. Results from 2–5 independent experiments are reported. The incubation time for all the tests was 4 hours. Bar show mean ± SEM. Statistical significance: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Mann–Whitney test was used to calculate statistical differences.

NKCEs override HLA-I inhibitory interactions. Degranulation activity of NK cells (All NK, gating on CD3−CD56+) and different NK-cell subsets [single (s)KIR2DL1+, KIR2DL3+/KIR3DL1+, and KIR−NKG2A+, identified by appropriate gating strategy reported in Supplementary Fig. S1] from healthy donors (n = 3) or transplanted patient (n = 1) upon 4-hour coculture with MHH-CALL-4 cells in presence of IC-NKp46- or CD19-NKp46-NKCE at 100 μg/mL. Results from three independent experiments are reported. A, Contour plot of a representative donor showing the degranulation of different NK-cell subsets in the presence of the target cells and NKCEs. Numbers indicate the percentage of cells in each quadrant. B, Data from 3 healthy donors are reported. C and D, Degranulation of NK-cell subsets from 1 patient 1-year after haplo-HSCT. C, Contour plot of flow cytometry (raw data). D, Graphical representation.

The long story of NK cells started about 50 y ago with the first demonstration of a natural cytotoxic activity within an undefined subset of circulating leukocytes, has involved an ever-growing number of researchers, fascinated by the apparently easy-to-reach aim of getting a “universal anti-tumor immune tool”. In fact, in spite of the impressive progress obtained in the first decades, these cells proved far more complex than expected and, paradoxically, the accumulating findings have continuously moved forward the attainment of a complete control of their function for immunotherapy. The refined studies of these latter years have indicated that NK cells can epigenetically calibrate their functional potential, in response to specific environmental contexts, giving rise to extraordinarily variegated subpopulations, comprehensive of memory-like cells, tissue-resident cells, or cells in various differentiation stages, or distinct functional states. In addition, NK cells can adapt their activity in response to a complex body of signals, spanning from the interaction with either suppressive or stimulating cells (myeloid-derived suppressor cells or dendritic cells, respectively) to the engagement of various receptors (specific for immune checkpoints, cytokines, tumor/viral ligands, or mediating antibody-dependent cell-mediated cytotoxicity). According to this picture, the idea of an easy and generalized exploitation of NK cells is changing, and the way is opening toward new carefully designed, combined and personalized therapeutic strategies, also based on the use of genetically modified NK cells and stimuli capable of strengthening and redirecting their effector functions against cancer.

ABSTRACT:TCRαβ/CD19 cell depletion is a promising graft manipulation technique frequently used in the context of human leukocyte antigen (HLA)-haploidentical hematopoietic stem cell transplantation (HSCT). We previously reported the results of a phase I-II clinical trial (NCT01810120) to assess the safety and the efficacy of this type of exvivo T-cell depletion in 80 children with acute leukemia, showing promising survival outcomes. We now report an updated analysis on a cohort of 213 children with a longer follow-up (median, 47.6 months for surviving patients). With a 5-year cumulative incidence of nonrelapse mortality of 5.2% (95% confidence interval [CI], 2.8%-8.8%) and a cumulative incidence of relapse of 22.7% (95% CI, 16.9%-29.2%), projected 10-year overall and disease-free survival (DFS) were 75.4% (95% CI, 68.6%-80.9%) and 71.6% (95% CI, 64.4%-77.6%), respectively. Cumulative incidence of both grade II-IV acute and chronic graft-versus-host disease were low (14.7% and 8.1%, respectively). In a multivariable analysis for DFS including type of disease, use of total body irradiation in the conditioning regimen (hazard ratio [HR], 0.5; 95% CI, 0.26-0.98; P = .04), disease status at HSCT (complete remission [CR] ≥3 vs CR 1/2; HR, 2.23; 95% CI, 1.20-4.16; P = .01), and high levels of pre-HSCT minimal residual disease (HR, 2.09; 95% CI, 1.01-4.33; P = .04) were independently associated with outcome. In summary, besides confirming the good outcome results already reported (which are almost superimposable on those of transplant from HLA-matched donors), this clinical update allows the identification of patients at higher risk of treatment failure for whom personalized approaches, aimed at reducing the risk of relapse, are warranted.

Supplementary Figure S7 shows distribution of HLA-B supertypes expressed by LCLs clustered in three groups based on MHC class I-fold changes

Table S2. Expression of TIGIT ligands in B cells and T cells.

<p>Supplementary Figure S5 shows surface expression of ligands for activating NK cell receptors on DAOY cells treated with ERAP1 inhibitor</p>

Supplementary Figure 3 from Natural Killer Cells Efficiently Reject Lymphoma Silenced for the Endoplasmic Reticulum Aminopeptidase Associated with Antigen Processing

Supplementary Figure S1 shows ERAP1 expression in HeLa cells transduced with different ERAP1 shRNA constructs

Supplementary Figure 1 from Natural Killer Cells Efficiently Reject Lymphoma Silenced for the Endoplasmic Reticulum Aminopeptidase Associated with Antigen Processing

Natural killer (NK) cell-based adoptive immunotherapy in leukemia patients is an emerging field of interest based on clinical evidence of efficacy and safety. Elderly acute myeloid leukemia (AML) patients have been successfully treated with NK cells from HLA-haploidentical donors, especially when high amounts of alloreactive NK cells were infused. The aim of this study was comparing two approaches to define the size of alloreactive NK cells in haploidentical donors for AML patients recruited in two clinical trials with the acronym “NK-AML” (NCT03955848), and “MRD-NK”. The standard methodology was based on the frequency of NK cell clones capable of lysing the related patient-derived cells. The alternative approach consisted of the phenotypic identification of freshly derived NK cells expressing, as inhibitory receptors, only the inhibitory KIR(s) specific for the mismatched KIR-Ligand(s) (HLA-C1, HLA-C2, HLA-Bw4). However, in KIR2DS2+ donors and HLA-C1+ patients, the unavailability of reagents staining only the inhibitory counterpart (KIR2DL2/L3) may lead to an underestimated identification of the alloreactive NK cell subset. Conversely, in the case of HLA-C1 mismatch, the alloreactive NK cell subset could be overestimated due to the ability of KIR2DL2/L3 to recognize with low-affinity also HLA-C2. Especially in this context, the additional exclusion of LIR1-expressing cells might be relevant to refine the size of the alloreactive NK cell subset. We could also associate degranulation assays, using as effector cells IL-2 activated donor peripheral blood mononuclear cells (PBMC) or NK cells upon co-culture with the related patient target cells. The donor alloreactive NK cell subset always displayed the highest functional activity, confirming its identification accuracy by flow cytometry. Despite the phenotypic limitations and considering the proposed corrective actions, a good correlation was shown by the comparison of the two investigated approaches. In addition, the characterization of receptor expression on a fraction of NK cell clones revealed expected but also few unexpected patterns. Thus, in most instances, the quantification of phenotypically defined alloreactive NK cells from PBMC can provide data similar to the analysis of lytic clones, with several advantages, such as a shorter time to achieve the results and, perhaps, higher reproducibility/feasibility in many laboratories.

Supplementary Methods, Legends for Figures 1-5 from Natural Killer Cells Efficiently Reject Lymphoma Silenced for the Endoplasmic Reticulum Aminopeptidase Associated with Antigen Processing

<p>Supplementary Figure S4 shows the gating strategy used to analyse degranulation of NK-cell subsets by flow cytometry</p>

Supplementary Figure 5 from Natural Killer Cells Efficiently Reject Lymphoma Silenced for the Endoplasmic Reticulum Aminopeptidase Associated with Antigen Processing

Supplementary Figure 1. IL-21 produc􀆟on is reduced in CD4 FL T cells; Supplementary Figure 2. TCR-induced signaling effectors have different kine􀆟c; Supplementary Figure 3. Iden􀆟fica􀆟on of T-cell subsets; Supplementary Figure 4. Distribu􀆟on of T-cell subsets in FL LN; Supplementary Figure 5. Expression pa􀆩erns of co-inhibitory receptors in CD8 and CD4 T-cell subsets; Supplementary Figure 6. Contribu􀆟on of Tregs among CD4+TIGIT+ T cells; Supplementary Figure 7. TIGIT is a highly expressed co-inhibitory receptor in FL; Supplementary Figure 8. TIGIT expression is stable over 􀆟me; Supplementary Figure 9. Recovery of TCR-induced signaling in TIGIT+ CD8+ FL T cells is robust.