Haploidentical natural killer cells induce remissions in non-Hodgkin lymphoma patients with low levels of immune-suppressor cells

Abstract

We report a novel phase 2 clinical trial in patients with poor prognosis refractory non-Hodgkin lymphoma (NHL) testing the efficacy of haploidentical donor natural killer (NK) cell therapy (NK dose 0.5-3.27 × 10⁷ NK cells/kg) with rituximab and IL-2 (clinicaltrials.gov NCT01181258). Therapy was tolerated without graft-versus-host disease, cytokine release syndrome, or neurotoxicity. Of 14 evaluable patients, 4 had objective responses (29%; 95% CI 12-55%) at 2 months: 2 had complete response lasting 3 and 9 months. Circulating donor NK cells persisted for at least 7 days after infusion at the level 0.6-16 donor NK cells/µl or 0.35-90% of total CD56 cells. Responding patients had lower levels of circulating host-derived Tregs (17 ± 4 vs. 307 ± 152 cells/µL; p = 0.008) and myeloid-derived suppressor cells at baseline (6.6 ± 1.4% vs. 13.0 ± 2.7%; p = 0.06) than non-responding patients. Lower circulating Tregs correlated with low serum levels of IL-10 (R ² = 0.64; p < 0.003; n = 11), suggestive of less immunosuppressive milieu. Low expression of PD-1 on recipient T cells before therapy was associated with response. Endogenous IL-15 levels were higher in responders than non-responding patients at the day of NK cell infusion (mean ± SEM: 30 ± 4; n = 4 vs. 19.0 ± 4.0 pg/ml; n = 8; p = 0.02) and correlated with day 14 NK cytotoxicity as measured by expression of CD107a (R ² = 0.74; p = 0.0009; n = 12). In summary, our observations support development of donor NK cellular therapies for advanced NHL as a strategy to overcome chemoresistance. Therapeutic efficacy may be further improved through disruption of the immunosuppressive environment and infusion of exogenous IL-15.

Keywords: Adoptive transfer NK cells; Cellular therapy; Chemotherapy-refractory non-Hodgkin lymphoma; IL-2; Immunosuppressive environment.

Fig. 1

NK cell phenotype and function in patients with relapsed/refractory lymphoma as compared to newly diagnosed NHL patients and healthy controls. a PBMC from NHL patients (n = 14) and healthy controls (n = 8) were rested overnight in medium and stimulated with target cells (K562, 20:1) and cytokines 6 h prior to staining. NK cell degranulation (CD107a), IFNγ production, and frequency of CD16-expressing NK cells were evaluated by flow cytometry. b Overnight-rested PBMCs from newly diagnosed (n = 4) or refractory NHL (8–14) and healthy controls (n = 6–7) were stained for TIGIT and TIM3 and evaluated by flow cytometry. Data are shown as mean ± SEM, and statistical analyses were done on pooled data using the Student’s unpaired t test. Each symbol represents an individual donor. New NHL newly diagnosed NHL

Fig. 2

Donor NK cells’ persistence. a Donor NK cell expansion in peripheral blood at day 7 depicted in histograms showing donor vs. recipient HLA gated on CD56⁺CD3⁻ cells. All 7 subjects with differentiating donor/recipient HLA antigens (A2 or A24) are shown. Patients 1, 2 and 3 were responders; patients 4, 5 and 6 were non-responders and patient 7 died from neutropenic sepsis at day 10 after NK cell infusion. Absolute lymphocyte cell (ALC) in cells/µL and whole blood standard very short tandem repeat (WB VNTR) at day 7 after NK cell infusion is shown for each patient. b Donors’ and recipients’ CD16 expression 7 days following infusion are shown as mean ± SEM, and statistical analysis were done on pooled data using the Student’s unpaired t test

Fig. 3

Correlation of response with endogenous IL-15. a PBMC from NHL patients (n = 14) and healthy controls (n = 7) were rested overnight in medium and evaluated for proliferation (Ki67) by flow cytometry. b Endogenous IL-15 serum levels were evaluated at different time points in NHL patients (n = 12) comparing responders and non-responders. Data are shown as mean ± SEM, and statistical analyses were done on pooled data using the Student’s unpaired t test. Each symbol represents an individual donor. c Correlation analyses (n = 12) evaluating the relationship between NK cell degranulation and endogenous IL-15 level after therapy (day 14). p value was calculated on adjusted slope

Fig. 4

Expression of suppressive receptors on circulating T cells. a PBMC from NHL patients were rested overnight in medium and then staining for PD-1 and TIGIT and evaluated by flow cytometry. Representative histograms showing PD-1 and TIGIT expression in bulk T cells (CD3) in responders vs. non-responders before (upper panel) and 14 days after treatment (lower panel). b Different T cell subsets (CD4, and CD8) were evaluated for PD-1 and TIGIT expression in responders (R) and non-responders (NR). Data are shown as mean ± SEM, and statistical analyses were done on pooled data using the Student’s unpaired t test. Each symbol represents an individual donor

Fig. 5

Circulating MDSC and regulatory T cell correlate with clinical response and NK cell proliferation. Circulating regulatory T cells and MDSC in NHL patients before and after therapy comparing responders (n = 4) and non-responders (n = 8–10). a, b PBMCs from NHL patients were rested overnight and stained, and then the frequencies of MDSCs and Tregs were determined by flow cytometry. Each symbol represents an individual donor. c, d Correlation analyses (n = 12) evaluating the relationship between NK cell proliferation and the numbers and frequency of Tregs and MDSCs in patients with NHL before and 14 days after treatment. Statistical analyses were done using Pearson correlation