CALGB 150905 (Alliance): rituximab broadens the antilymphoma response by activating unlicensed NK cells

Abstract

Natural killer (NK) cells contribute to clinical responses in patients treated with rituximab, but the rules determining NK-cell responsiveness to mAb therapies are poorly defined. A deeper understanding of the mechanisms responsible for antibody-dependent cellular cytotoxicity (ADCC) could yield useful biomarkers for predicting clinical responses in patients. Unlicensed NK cells, defined as NK cells lacking expression of an inhibitory KIR for self-HLA class I ligands, are hyporesponsive in steady state, but are potent effectors in inflammatory conditions. We hypothesized that antitumor antibodies such as rituximab can overcome NK-cell dependence on licensing, making unlicensed NK cells important for clinical responses. Here, we examined the influences of variations in KIR and HLA class I alleles on in vitro responses to rituximab. We tested the clinical significance in a cohort of patients with follicular lymphoma treated with rituximab-containing mAb combinations, and show that rituximab triggers responses from all NK-cell populations regardless of licensing. Neither IL2 nor accessory cells are required for activating unlicensed NK cells, but both can augment rituximab-mediated ADCC. Moreover, in 101 patients with follicular lymphoma treated with rituximab-containing mAb combinations, a "missing ligand" genotype (predictive of unlicensed NK cells) is associated with a higher rate of progression-free survival. Our data suggest that the clinical efficacy of rituximab may be driven, in part, by its ability to broaden the NK-cell repertoire to include previously hyporesponsive, unlicensed NK cells. A "missing ligand" KIR and HLA class I genotype may be predictive of this benefit and useful for personalizing treatment decisions in lymphomas and other tumors.

Figure 1. Rituximab triggers degranulation and IFNγ release from hypo-responsive KIR-NKG2A⁻ and unlicensed spKIR⁺NKG2A⁻ NK cells

(A) The vertical axis depicts the percentage of NK cells expressing CD107a (a marker of degranulation) after 4 hours of incubation with CD20⁺ B-cell lymphoblasts (721.221) +/− rituximab. Identification of degranulation by discrete NK cell subsets was performed as shown in Figure 1, using freshly isolated, resting PBMCs from subject # 1 (HLA-C2/C2, Bw4/Bw6). The KIR⁻NKG2A⁻ and unlicensed spKIR2DL3⁺NKG2A⁻NK cell subsets responded poorly to CD20⁺ lymphoblasts alone, but coating the lymphoblasts with 10 ug/ml of rituximab for 30 minutes activated these hypo-responsive NK cell subsets. (B) Intracellular IFNγ responses between KIR⁻NKG2A⁻ and unlicensed spKIR2DL1⁺NKG2A⁻ NK cells from subject #2 (HLA-C1/C1, Bw4/Bw6) were consistent with the degranulation responses, with a 2–3-fold increase in the percentage of hypo-responsive, KIR⁻NKG2A⁻ and spKIR2DL1⁺NKG2A⁻ NK cell subsets releasing IFNγ after the addition of rituximab. (C) Aggregate degranulation responses of NK cells exclusively expressing an unlicensed KIR, licensed KIR, or KIR⁻NKG2A⁻ from 8 subjects with distinct KIR - HLA genotypes and circulating NK cell repertoires. Data were combined into three functional groups: NK cells lacking KIR2DL1, 2DL2, 2DL3, 3DL1, and NKG2A (KIR⁻NKG2A⁻); NK cells expressing unlicensed KIR (spKIR3DL1⁺HLA-Bw6/6, KIR2DL1⁺HLA-C1/C1, or KIR2DL2/3⁺HLA-C2/C2); and NK cells expressing licensed KIR (spKIR3DL1⁺HLA-Bw4, KIR2DL1⁺HLA-C2, or KIR2DL2/3-HLA-C1). Statistical comparisons were made using the Wilcoxon test, with **P<0.01. In subjects with more than 1 unlicensed or licensed KIR⁺ NK cell subset, the dot represents the total number of CD107a⁺ spKIR NK cells divided by the number of spKIR NK cells. Experiments were performed in triplicates; horizontal bar represents mean, with standard errors of mean.

Figure 2. Coating Raji B-cell lymphoblast with rituximab overcomes intrinsic NK cell tolerance of the unlicensed spKIR3DL1⁺NKG2A⁻, KIR⁻NKG2A⁻ and hypo-responsive spKIR3DL2⁺NKG2A⁻ NK cell subsets

Percentage of NK cells expressing cell surface CD107a exclusively expressing spKIR3DL1, spKIR3DL2, and NK cells lacking inhibitory KIR expression for self-HLA using subject 11 (HLA-C1/C2, Bw6/6, A11/A31) after 4 hours of incubation with a different CD20-expressing B-lymphoblast cell line (Raji) confirmed to match the HLA-B KIR ligand status of subject 11. Unlicensed spKIR3DL1⁺NKG2A⁻, spKIR3DL2⁺NKG2A⁻, and KIR⁻NKG2A⁻ NK cells have minimal “missing self” activity against CD20⁺ HLA-class I-deficient 721.221 and HLA-Bw6/6 Raji B-lymphoblasts. Incubating both CD20⁺ B-lymphoblasts with 10ug/ml of rituximab stimulates robust degranulation from these hypo-responsive unlicensed NK cell subsets.

Figure 3. Rituximab alone is sufficient to activate unlicensed NK cells, which is augmented by IL-2 and higher doses of rituximab

(A) To determine whether cytokines secreted from other mononuclear cells within the PBMC population are required for rituximab-dependent activation of unlicensed NK cells, NK cells from subject #8 (HLA-C1/C1, Bw6/Bw6) were purified by antibody-coated magnetic bead sorting and incubated for 4 hours with rituximab-coated 721.221 B-lymphoblasts. The CD107a response of NK cell subsets is shown; similar results were seen using subject #2 (data not shown). (B) To evaluate requirements for IL-2, PBMCs from subjects #2, #1, #8, and #11 were incubated overnight with or without 1,000 U/ml of IL-2 prior to co-culture with rituximab-coated Raji cell lines. (C) Escalation of rituximab concentration demonstrates dose-dependent activation of unlicensed NK cells (subject #17). (D) NK cells from 3 study subjects (#17, #18, and #19) were stimulated in vitro with increasing concentrations of rituximab and a fixed NK cell to 721.221 cell ratio of 1:2. CD107a was measured on individual NK cell subsets and licensing was determined based on KIR expression and HLA class I genotyping. Statistical significance was defined as p<0.05. Experiments were performed in duplicates for each study subject.

Figure 4. Hypo-responsive NK cells exclusively expressing KIR3DL2 are activated by rituximab

Shown is the degranulation (CD107a) response by NK cells exclusively expressing KIR3DL2 induced by 721.221 B-lymphoblasts with or without rituximab. CD107a results from 4 subjects were aggregated: subject #11 (HLA-A11/31), #6 (HLA-A11/02), #4 (HLA-A3/26), and #3 (HLA-A02/02). Similar to the KIR⁻NKG2A⁻ (shown) and unlicensed spKIR⁺NKG2A⁻ (not shown) NK cells, NK cells exclusively expressing KIR3DL2 were hypo-responsive to 721.221 B-lymphoblast tumor cells alone, but degranulated after the addition of rituximab, comparable to the licensed NK cell subset. Statistical comparisons were made using the Mann-Whitney and Kruskall-Wallis tests, with *P<0.05. The presence of the HLA-A11 (subject #11, #6) or -A3 (subject #4) ligand did not influence the antibody-dependent activation of the spKIR3DL2⁺ NK cell subset.

Figure 5. Influence of a “missing ligand” genotype on PFS in follicular lymphoma patients treated with rituximab antibody combinations

(A) Progression-free survival among FL patients treated with rituximab-containing monoclonal antibody combinations, and stratified according to: (B) the presence or absence of HLA-A11, (C) missing the HLA-C2 ligand for KIR2DL1, (D) missing the HLA-Bw4 ligand for KIR3DL1, and (E) the presence or absence of both HLA-C2 and HLA-Bw4 ligands.