Exercise-mobilized lymphocytes enhance antibody-based immunotherapy in multiple myeloma through CD16⁺ NK cell–mediated cytotoxicity

Abstract

Background

Immunotherapies have significantly improved treatment outcomes in multiple myeloma (MM); however, challenges remain due to variable efficacy, toxicities, and off-target effects. Exercise-induced lymphocyte mobilization presents a novel adjunctive strategy to enhance immune-based cancer therapies. This study aimed to determine if lymphocytes mobilized by acute exercise could synergize with established MM therapeutic regimens to improve anti-myeloma cytotoxicity.

Methods

We used an ex vivo model to assess the cytotoxicity of resting and exercise-mobilized lymphocytes against drug-sensitive (MM1.S) and resistant (MM1.R) MM cell lines in combination with established MM therapeutic regimens—lenalidomide and dexamethasone combined with either daratumumab (DRd) or magrolimab (MRd). Blood lymphocytes were collected from twenty-two healthy participants at rest and during 20-minutes of graded cycling exercise. An ex vivo approach was used to model two clinically relevant scenarios: (1) a pre-treatment approach prior to exercise-mobilized donor lymphocyte infusion (DLI-X) and (2) a co-treatment approach mimicking exercise during ongoing therapy.

Results

Exercise-mobilized lymphocytes demonstrated enhanced cytotoxicity against both drug-sensitive (MM1.S: ≤ 2.3-fold) and resistant (MM1.R: ≤ 2.4-fold) cell lines, which was further augmented by DRd or MRd (≤ 2.4-fold for MM1.S and ≤ 1.9-fold for MM1.R). In the co-treatment setting, NK-cells purified during exercise were more effective than resting NK-cells at mediating antibody-dependent cellular cytotoxicity (ADCC) with daratumumab (≤ 1.6-fold increase) and magrolimab (≤ 1.2-fold increase). This effect reflected the preferential mobilization of CD16⁺ NK-cells (≤ 6.83-fold from rest), as CD16 blockade substantially diminished the exercise-induced ADCC enhancement (≤ 6.3-fold reduction). Cytotoxicity was greater in MRd versus DRd, likely reflecting daratumumab-induced fratricide of CD38⁺ NK-cells (≤ 1.4-fold decrease), which was particularly evident among exercise-mobilized NK-cells.

Conclusion

These findings demonstrate that acute exercise mobilizes a potent NK cell subset that enhances monoclonal antibody efficacy against myeloma cells. These findings also highlight the potential for carefully timed and context-specific integration of exercise to optimize monoclonal antibody efficacy in MM.

Trial registration

This study was conducted as part of the ‘Exercise as an Immune Adjuvant for Allogeneic Cell Therapies (Allo-X)’ trial, registered on 2024-10-16 at ClinicalTrials.gov, NCT06643221.

Supplementary information

The online version contains supplementary material available at 10.1186/s12967-026-07888-7.

Background

Exercise has emerged as a potent non-pharmacological adjuvant in oncology, shown to reduce cancer incidence and improve quality and duration of life in cancer survivors [1–3]. In parallel, immunotherapies such as adoptive cell transfer (ACT), monoclonal antibodies, and immune checkpoint inhibitors have transformed cancer treatment [4]. Increasing interest now focuses on whether exercise can augment the efficacy of these immune-based therapies [5, 6]. A key mechanism underlying this synergy is the acute mobilization and redistribution of effector lymphocytes such as natural killer (NK cells, CD8⁺ T cells, and γδ T cells in response to exercise [7, 8]. This catecholamine-driven response mobilizes cells with enhanced anti-tumor phenotypes and transcriptional profiles, promoting increased tumor infiltration and growth suppression in multiple murine cancer models as well as cancer patients [9–15].

Immunotherapies play a central role in the treatment of hematologic malignancies, particularly multiple myeloma (MM)—an incurable disease accounting for ~10% of all hematologic cancers and characterized by the proliferation of malignant plasma cells in the bone marrow [16–18] (Dima et al., 2022; Pinto et al., 2020; Rajkumar & Kumar, 2020). Over the past two decades, the integration of autologous stem cell transplantation (ASCT) with monoclonal antibodies (e.g., daratumumab), immunomodulatory drugs (e.g., lenalidomide), and corticosteroids has significantly improved outcomes [16–18]. More recently, allogeneic hematopoietic cell transplantation (alloHCT), donor lymphocyte infusion (DLI), and autologous CAR T-cell therapies have shown promise in MM, but remain limited by variable efficacy, treatment-related mortality, and distinct toxicities. Allogeneic approaches are further complicated by risks of graft-versus-host disease (GVHD), while both allogeneic and autologous cell therapies face challenges including limited persistence, antigen escape, cytokine release syndrome and neurotoxicity [19–23]. Most patients ultimately develop multi-drug resistance and relapse, underscoring the urgent need for strategies that can enhance efficacy while minimizing toxicity [24].

Exercise-induced immunomodulation offers a promising approach to address these gaps. Exercise preferentially mobilizes CD16⁺ NK-cells—key mediators of antibody-dependent cellular cytotoxicity (ADCC)—and has been shown to enhance daratumumab-mediated cytotoxicity in MM patients [25, 26]. However, in patients receiving daratumumab, the benefits of exercise may be constrained due to the preferential mobilization of CD38+ NK-cells, which are also targeted and depleted by daratumumab through ADCC [26, 27]. To mitigate this, monoclonal antibodies less likely to cause NK-cell fratricide, such as magrolimab (anti-CD47), may offer improved synergy with exercise [28]. In addition, exercise could be leveraged not by the patient, but by the donor, allowing us to harvest exercise-mobilized lymphocytes for use in DLI (DLI-X) [29]. This strategy circumvents prior drug exposure and allows timing of DLI-X after standard therapy to maximize ADCC potential. Preclinical data from our group demonstrate that DLI-X enhances anti-leukemia activity while reducing GVHD risk in xenogeneic mouse models [13, 29].

The overarching aim of this study was to evaluate the synergistic potential of acute exercise with established MM therapies—specifically lenalidomide and dexamethasone combined with either daratumumab (DRd) or magrolimab (MRd)—across two clinically relevant scenarios. The first simulated a pre-treatment approach in which DRd or MRd is followed by ACT using DLI-X. The second modeled a co-treatment scenario in which MM patients engage in acute exercise during ongoing DRd or MRd therapy. We found that exercise-mobilized lymphocytes exhibit enhanced cytotoxicity against both drug-sensitive (MM1.S) and drug-resistant (MM1.R) myeloma cells and greater synergy with MM combination therapies. Additionally, we revealed that these effects are primarily driven by the increased mobilization of NK-cells and identified that the enhanced CD16 expression on NK-cells after exercise is a key mechanism in the response. These results suggest that exercise may be a feasible and effective adjuvant to enhance immune-based therapies in MM.

Methods

Participants

Twenty-two healthy participants completed this study (Table 1) and were recruited on our registered clinical trial, entitled ‘Exercise as an Immune Adjuvant for Allogeneic Cell Therapies (Allo-X)’ (ClinicalTrials.gov ID NCT06643221). Study participants were not taking medications (except for oral contraceptives for some female participants) and were classified as “low risk” for graded exercise testing according to ACSM/American Heart Association criteria. Participants abstained from alcohol, caffeine, and physical activity for 24 hours before each laboratory visit. All participants provided written informed consent, and the study was approved by the Human Subjects Protection Program at the University of Arizona on protocol #17–338. All laboratory visits and exercise trials were conducted between 6:00am-9:00am local time.

Table 1.

Physical and demographic characteristics of the participants (n = 22; 14 males and 8 females) included from the ‘allo-X’ trial (NCT06643221)

Variable	Mean	SD	Range
Age (Years)	31.9	10.02	(21–51)
Height (cm)	176.5	12.9	(155–193.04)
Weight (kg)	79.8	18.3	(52.2–130.8)
Body Mass Index (kg m−2)	25.3	3.2	(21.42–36.04)
VO2max (ml kg−1 min−1)	43.2	9.0	(26.81–65.06)
VO2max Rating	3.5	1.5	(1–6)

VO_2max Rating determined by assigning a numerical core of 1 (‘very poor’) to 6 (‘superior’) in accordance with ACSM age and sex-adjusted score categories of cardiorespiratory fitness. cm, centimeter; kg, kilogram. BMI=body mass index

Exercise trial design

The protocol was conducted as previously described in detail [30]. In brief, participants performed a maximal graded exercise test on an Excalibur Sport cycle ergometer (Lode B.V., Groningen, The Netherlands), with respiratory gases analyzed by a Quark CPET metabolic cart (COSMED S.R.L., Rome, Italy) to determine maximal oxygen uptake (VO_2max) and establish workloads for the main trial. During the subsequent (main) trial, an indwelling catheter was inserted into an antecubital vein for serial blood sampling at rest, during exercise (end of the 80% VO_₂max stage), and 1 h post exercise (+1 h). Following a 5-min 50 W warm-up, participants then performed a 20-minute continuous cycling protocol consisting of four, 5-minute incremental stages at 50, 60, 70, and 80% VO2max, maintaining ≥60 rpm; heart rate, VO₂, and rating of perceived exertion (RPE) were monitored continuously. Blood was collected into ACD tubes for Peripheral blood mononuclear cells (PBMCs) isolation and K₂EDTA tubes for whole-blood phenotyping. The experimental design id depicted in Figure 1A.

Fig. 1.

Schematic detailing the experimental design. The workflow illustrates the collection of lymphocytes from participants, followed by ex vivo co-culture with myeloma cells under pre-treatment and co-treatment conditions with therapeutic regimens. (A) Overview of the clinical exercise protocol. Participants completed a screening visit followed by the main exercise trial. Peripheral blood was collected at rest, during, and 1-hour post-exercise to isolate and cryopreserve PBMCs for subsequent analysis. (B) Ex vivo treatment and co-culture workflow. Effector cells (PBMCs or isolated NK cells) and target cells (MM1.S and MM1.R) were incubated overnight under specific conditions. Following incubation and washout, effectors and targets were co-cultured to assess cytotoxicity (with or without CD16 blockade), immune phenotype, and tumor ligand expression. Abbreviations: DRd: Daratumumab, lenalidomide, and Dexamethasone; MRd: Magrolimab, lenalidomide, and Dexamethasone

Blood sample analysis and processing

Complete blood counts were immediately performed on whole blood samples treated with EDTA using an automated hematology analyzer (DxH 560 Autoloader Hematology Analyzer, Beckman Coulter). Whole blood samples were labeled with directly conjugated antibodies for multi-parameter flow cytometry to enumerate CD45⁺CD14⁺ monocytes and CD45⁺CD14^- lymphocytes subsets, as previously described [30]. Briefly, 100 µL of EDTA whole blood was incubated with the following antibodies CD8-VioBlue, CD57-VioBlue, CD14-VioGreen, CD3-VioGreen, CD62L-FITC, NKG2C-FITC, TCR-Vd2-FITC, CD4-PE, NKG2D-PE, CD45-PE-Vio615, CD20-Pe-Vio770, CD45RA-PerCPVio770, NKG2A-Pe-Vio770, CD16-APC, KLRG1-APC, and CD56-APC-Vio770 (Miltenyi Biotec Inc., Gernany) for 30 min at room temperature. Samples were lysed with RBC lysis buffer (Miltenyi Biotec Inc., Gernany) for 20 min at room temperature and washed three cycles before being analyzed using a MACSQuant 14 flow cytometer (Miltenyi Biotec Inc., Germany). The total cell numbers of each lymphocyte subset were determined by multi-parameter flow cytometry by multiplying the percentage of all lymphocytes expressing the surface markers of interest by the total lymphocyte count. PBMCs were purified from whole blood (Ficoll-Paque PLUS, Cytiva), then cryopreserved in liquid nitrogen at a concentration of 10 × 10⁶ cells/mL in freezing media (90% FBS, 10% DMSO) until they were used for cytotoxicity assays, phenotyping, and NK cell isolations.

Isolation of NK cells

PBMCs were thawed and counted using a MACSQuant 14 flow cytometer. NK-cells were then isolated from the PBMCs by negative selection according to the manufacturer’s protocol (Human NK Cell Isolation Kit, Miltenyi Biotec). Briefly, PBMCs were resuspended in an isolation buffer (PBS with 0.5% BSA and 2 mM EDTA) and incubated sequentially with the kit’s Biotin-Antibody Cocktail (5 min, 2–8°C) and MicroBead Cocktail (10 min, 2–8°C) before autoMACS® Pro separation (Miltenyi Biotec Inc., Germany). The purity of the isolated NK-cells was confirmed to be > 95% by flow cytometry. Finally, purified NK-cells were seeded at 0.5 × 10⁶ cells/mL in 24-well plates for overnight culture. To maintain NK cell viability during the overnight incubation period, the culture media (RPMI-1640 with 10% FBS and 1% penicillin-streptomycin) was supplemented with recombinant human IL-15 (500 IU/mL) and contained one of the following treatments: no treatment (control); DRd (Daratumumab 10 µg/mL, lenalidomide 2 µM, dexamethasone 50 nM); MRd (Magrolimab 10 µg/mL, lenalidomide 2 µM, dexamethasone 50 nM); Daratumumab (10 µg/mL); or Magrolimab (10 µg/mL). We note that the supplementation of IL-15 was identical for all experimental groups; thus, relative comparisons between resting and exercise conditions remain valid, reflecting the differential impact of the physical stressor rather than culture artifacts. Drug concentrations were selected to approximate clinically relevant pharmacokinetics reported in human plasma and prior in vitro literature [31–33]. Lenalidomide (2 µM) and dexamethasone (50 nM) were utilized at concentrations corresponding to therapeutic plasma levels observed in patients following standard dosing regimens. For monoclonal antibodies, daratumumab and magrolimab were used at 10 µg/mL, a concentration sufficient to saturate cell surface receptors and induce maximal ADCC in vitro [34].

Surface and intracellular staining on lymphocytes and tumors

Frozen PBMCs were thawed and pretreated for 24 h with daratumumab or magrolimab (10 µg/mL; Selleckchem, USA), lenalidomide (2 µM; Selleckchem, USA), and dexamethasone (50 nM). Cells were then stained for 15–20 min at room temperature with fluorochrome-conjugated antibodies: CD8-VioBlue, CD57-VioBlue, CD3-VioGreen, CD47-FITC, NKG2C-FITC, CD4-PE, NKG2D-PE, CD45-PE-Vio615, CD16-PerCP-Vio700, CD32-PE-Vio770, CD38-APC, DNAM-1-APC, and CD56-APC-Vio770 (Miltenyi Biotec, Germany). After washing, samples were resuspended in 300 µL PBS and acquired using a MACSQuant 14 flow cytometer. Similarly, MM1.S tumor cells were pretreated for 24 h and stained with antibodies against MICA/MICB, CD155, PD-L1, PD-L2, HLA-DR/DP/DQ, CD38, CD47, and Fas (Miltenyi Biotec); ULBP-1, ULBP-3, ULBP2/5/6 (R&D Systems, USA); Nectin-2 and HLA-E (BioLegend, USA). Data were acquired on a BD LSRFortessa™ flow cytometer (BD Biosciences, USA).

Cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) blocking assay

The functionality of PBMCs and isolated NK-cells was assessed using a 4-hour flow cytometry-based cytotoxicity assay under three conditions (1): Control: targets and effectors untreated (2); Pre-treatment: targets only exposed to drugs overnight; effectors untreated; and (3) Co-treatment: both targets and effectors exposed to drugs overnight. For all treatment conditions, cells were washed free of drugs prior to co-culture. The therapeutic regimens investigated were DRd (Daratumumab 10 µg/mL, lenalidomide 2 µM, dexamethasone 50 nM), MRd (Magrolimab 10 µg/mL, lenalidomide 2 µM, dexamethasone 50 nM), Daratumumab (10 µg/mL), or Magrolimab (10 µg/mL). All effector cell cultures were supplemented with IL-15 (500 IU/mL) to support activation and survival. Following overnight culture, cells were prepared for the assay. Target cells were washed and labeled with an anti-CD71 antibody (Miltenyi Biotec Inc., Germany). For ADCC blocking experiments, isolated NK effector cells were incubated with an anti-human CD16 antibody (Clone 3G8; STEMCELL Technologies) or an IgG control for 30 minutes at room temperature. Effector cells were counted using a MACSQuant 14 flow cytometer. The experiemtal design for the cytotoxicity and ADCC assays is depicted in Fig. 1B.

Effector and labeled target cells were then co-cultured in duplicate U-bottom 96-well plates. PBMCs were plated at effector-to-target (E:T) ratios of 1.25:1, 2.5:1, 5:1, and 10:1; isolated NK-cells were plated at a fixed 2:1 ratio. A target-only condition (0:1 E:T ratio) was included to measure spontaneous lysis. After a 4-hour incubation (37 °C, 5% CO2), propidium iodide (PI; Thermo Fisher Scientific) was added to each well, and samples were immediately analyzed on a BD LSRFortessa™ Flow Cytometer. Cytotoxicity was determined by the percentage of dead target cells (CD71+PI+), and specific lysis was calculated as: Specific Lysis (%) =(%Total Lysis−%Spontaneous Lysis).

Statistical analysis

All statistical analyses were performed using GraphPad Prism (GraphPad Software, CA). Data are presented as mean ± SEM. All statistical tests were two-sided, and a p-value ≤0.05 was considered significant. The specific statistical models (e.g., repeated-measures ANOVA, paired t-tests) and any subsequent multiple-comparison procedures are specified in the relevant figure legends.

Results

Acute exercise induces dynamic changes in circulating immune cell subsets

A single bout of acute exercise induced a significant mobilization of monocytes and all major lymphocyte subsets (CD3⁺, CD4⁺, CD8⁺, B, γδ T, NK, and NK-T cells) (main effect of time, p < 0.001), which then either returned to baseline or dropped below resting levels during a 1-hour recovery period (Table 2). While total lymphocytes (+209±37%), monocytes (+112±46%), and various T cells all increased, the most substantial response was observed in NK-cells (+345±173%). This mobilization was even more pronounced for the ADCC-competent CD16⁺ NK cell subset (+396±187%), and both populations subsequently dropped below baseline at 1-hour post-exercise (−32±33% and −37±37%, respectively). The substantial and preferential mobilization of these highly cytotoxic CD16+ NK-cells supports the hypothesis that acute exercise could serve as a potent adjuvant strategy to enhance immune surveillance and the efficacy of antibody-based therapies.

Table 2.

Immune cell mobilization at rest, exercise, and 1-hour post-exercise with percentage mobilization and egress, and main effect from one-way repeated measures ANOVA. Data are mean ± SD, n = 22

(cells/µL)	Rest	Exercise	1-hr post-exercise	% Mobilization	% Egress	Main effect of time
Monocytes	372 ± 135.2	758.8 ± 208.6****	406.7 ± 99.37††††	112.1 ± 46.4	−45.3 ± 9.2	p < 0.0001 F(1.699, 35.68) = 121.3
Lymphocytes	1846 ± 468	3773 ± 778.1****	1582 ± 378.8**††††	109.2 ± 36.6	−57.6 ± 8.46	p < 0.0001 F(1.604, 33.68) = 227.1
NK cells	359.6 ± 172.9	1425 ± 499.6****	212.5 ± 101.7***††††	344.7 ± 172.8	−84 ± 7.5	p < 0.0001 F(1.120, 21.83) = 121.5
CD16+ NK cells	316.7 ± 172	1390 ± 501.4****	168.9 ± 89.45**††††	395.8 ± 187.1	−87.34 ± 5.2	p < 0.0001 F(1.135, 19.86) = 119.5
CD3+ cells	1185 ± 353.7	1866 ± 588.5****	1108 ± 315††††	58.99 ± 33.14	−38.8 ± 14.4	p < 0.0001 F(1.716, 33.46) = 50.17
CD4+ T cells	578.9 ± 190.2	750.6 ± 237.5***	603.1 ± 187.2†††	33 ± 31.8	−20.1 ± 17.3	p < 0.0001 F(1.880, 36.67) = 12.91
CD8+ T cells	412.3 ± 182.6	757.9 ± 344.4****	365 ± 144.7††††	92.53 ± 56.08	−32.9 ± 70.5	p < 0.0001 F(1.251, 24.39) = 42.80
CD4+ CD8+ T cells	56.06 ± 31.88	71.25 ± 35.59***	37.54 ± 23.93*†††	96.71 ± 39.33	−43.7 ± 25.8	p < 0.0001 F(1.699, 35.68) = 12.44
CD4- CD8- T cells	124.7 ± 80.94	252.8 ± 194.5	104.3 ± 66.11*†††	96.71 ± 39.33	7.6 ± 285.1	p = 0.0001 F(1.066, 20.78) = 21.96
CD3+ CD56+ cells	144.9 ± 93.75	334.4 ± 200.2****	108.1 ± 71.83*††††	153.4 ± 79.88	−64.9 ± 16.5	p < 0.0001 F(1.162, 22.67) = 38.16
γδ T cells	84.84 ± 100.4	159 ± 159.5**	70.86 ± 81.32††	223.3 ± 720.7	−15.0 ± 169.1	p = 0.0008 F(1.157, 21.40) = 13.95
B cells	210 ± 117.3	297.9 ± 126.5****	176.4 ± 80.68†††	52.87 ± 41.32	−38.2 ± 19.2	p < 0.0001 F(1.563, 30.47) = 19.16

*Indicates a significant difference from Rest at p < 0.05, **indicates a significant difference from Rest at p < 0.01, ***indicates a significant difference from Rest at p < 0.001, †indicates a significant difference from Exercise at p < 0.05, ††indicates a significant difference from Exercise at p < 0.01, †††indicates a significant difference from Exercise at p < 0.001 following Tukey multiple comparisons. ANOVA, analysis of variance. Mobilization= (Exercise cell count−Resting cell count)×100/Resting cell count Egress= (1-hr post-exercise cell count−Exercise cell count)×100/Exercise cell count

Exercise-mobilized lymphocytes exert potent anti-myeloma activity and synergize with multiple myeloma combination therapies

We assessed the cytotoxicity of exercise-mobilized lymphocytes, alone and with two MM regimens, using the schematic in Fig. 1B. Exercise preferentially mobilizes CD16⁺ NK-cells (Table 2), key mediators of ADCC, raising the question of whether this enhances anti-MM activity. Exercise-mobilized lymphocytes consistently exhibited significantly greater cytotoxicity than resting cells against both drug-sensitive (MM1.S) and resistant (MM1.R) lines (Fig. 2A–D). This increase translated into enhanced synergy with MM therapies, though outcomes depended on regimen and timing. With the DRd regimen, significant synergy was observed only with exercise-mobilized lymphocytes under pre-treatment conditions against both MM1.S and MM1.R (Fig. 2A, B). Under co-treatment, synergy was lost against MM1.S, likely due to daratumumab-induced NK cell fratricide. To address this, we tested MRd. Unlike DRd, MRd produced consistent synergy with exercise-mobilized lymphocytes in both pre- and co-treatment conditions against MM1.S (Fig. 2C). Results in MM1.R were mixed: MRd pre-treatment synergized with both resting and exercise lymphocytes, whereas co-treatment showed only a trend for resting cells (p = 0.0579) (Fig. 2D). Overall, MRd mitigated daratumumab-induced fratricide, making it a more favorable partner for exercise-mobilized lymphocytes.

Fig. 2.

Exercise-mobilized lymphocytes potentiate DRd/MRd-mediated killing of myeloma cells under pre- and co-treatment conditions. The graphs show the results of cytotoxicity assays under two conditions: pre-treatment where only target myeloma cells were exposed to the drug regimen overnight before co-culture with effector lymphocytes, and Co-treatment where both target myeloma and lymphocytes were exposed to drug regimen overnight before co-culture. (A–B) Cytotoxicity assays using the DRd (daratumumab + lenalidomide + dexamethasone) regimen against the multiple myeloma cell lines MM1.S (A) and MM1.R (B). (C–D) Cytotoxicity assays using the MRd (magrolimab + lenalidomide + dexamethasone) regimen against MM1.S (C) and MM1.R (D). Each curve displays the percentage of specific lysis across a range of effector-to-target (E:T) ratios. Red lines correspond to effector lymphocytes collected at rest (REST), while blue lines correspond to effectors collected during exercise at 80% VO_2max (EX). Solid lines with filled symbols represent conditions without the drug regimen, whereas dashed lines with open symbols represent conditions with the respective drug regimen (e.g., DRd-REST, DRd-EX). Data are presented as the mean ± SEM from N = 7 donors. Each condition was assayed in technical duplicate, with the values averaged for each donor. Statistical analysis was performed using a two-way repeated-measures ANOVA within each model, followed by Tukey’s multiple comparisons test. A solid horizontal bar denotes a significant exercise effect (EX vs. REST). A dashed horizontal bar denotes a significant treatment effect within a specific effector cell type (e.g., DRd-EX vs. EX). Significance is denoted as *p < 0.05 and **p < 0.01. “ns” indicates not significant. Abbreviations: DRd, daratumumab/lenalidomide/dexamethasone; MRd, magrolimab/lenalidomide/dexamethasone; E:T, effector-to-target ratio; SEM, standard error of the mean

Exercise-mobilized NK-cells enhance anti-myeloma activity through favorable phenotypic adaptations

Because DRd co-treatment impaired exercise effects, we examined whether exercise exacerbated NK cell fratricide by assessing CD38 and CD47 expression. Consistent with prior work (Collier-Bain et al., 2024), exercise-mobilized NK-cells displayed a higher percentage of CD38⁺ cells and elevated CD38 MFI compared with resting cells (p = 0.0139 and p = 0.0219), while CD47 levels were unchanged (Fig. 3A). A significant reduction in NK cell frequency was observed only in exercise groups treated with DRd (p = 0.0077), but not with MRd (Fig. 3B), implicating high CD38 expression in daratumumab-induced fratricide. Following overnight IL-15 culture, exercise-mobilized NK-cells expressed higher levels of cytotoxicity-associated markers—CD16, CD57, NKG2D, and DNAM-1 (all p < 0.01)—and lower CD32 (p = 0.0099) compared with resting cells (Fig. 3C–E). Two-way repeated-measures ANOVA across regimens revealed significant drug effects on CD16, CD57, NKG2D, NKG2C, DNAM-1, and CD38 MFI (all p < 0.001) (Fig. 3C–E). Post hoc testing showed that the exercise effect on CD16% was abolished by DRd but preserved under MRd, which yielded the highest CD16% overall (vs. exercise control, p = 0.0352; vs. exercise DRd, p = 0.0011). Although both regimens reduced CD16 MFI (p < 0.01), MRd preserved levels significantly better than DRd (p = 0.0016), with a trend toward higher expression in the exercise + MRd group (p = 0.0715). CD32% rose slightly with treatment, reaching significance only in rest + MRd (p = 0.0173). Increases in CD57 MFI, NKG2D%/MFI, and DNAM-1% in exercise-mobilized NK-cells were generally attenuated after drug exposure (Fig. 3D–E). Still, elevated CD57⁺ cell frequency persisted across both DRd and MRd (p < 0.02). Notably, only MRd maintained the exercise effect on NKG2D⁺ cells (p = 0.0373). DNAM-1 was differentially affected: DRd significantly reduced DNAM-1% compared with control in both rest and exercise conditions (p < 0.004), whereas MRd preserved it, showing trends toward higher DNAM-1 MFI versus DRd (rest, p = 0.0673; exercise, p = 0.0649). Finally, only MRd reduced NKG2C% relative to control (p < 0.002). Together, these findings indicate that MRd preserves the favorable phenotype of exercise-mobilized NK-cells, whereas DRd promotes fratricide and blunts their functional advantage.

Fig. 3.

Exercise-mobilized NK cells display enhanced cytotoxic phenotypes and differential responses to DRd vs MRd. (A) Baseline CD38% positive and MFI) and CD47% positive) on NK cells measured in peripheral blood mononuclear cells (PBMCs) collected at rest (REST; red) or during acute exercise at 80% VO2max (EX; blue). (B) NK-cell frequency (% of CD45+ lymphocytes) after overnight IL-15 culture under control, DRd (daratumumab + lenalidomide + dexamethasone), or MRd (magrolimab + lenalidomide + dexamethasone) conditions. Representative CD3 vs. CD56 dot plots are shown on the right for REST and EX across regimens, with the NK-cell gate (CD3−CD56+) indicated. (C) Expression of monoclonal-antibody–associated markers after overnight culture: CD38% positive and MFI) under control vs. MRd conditions, and CD47% positive and MFI) under control vs. DRd conditions. (D) Fc-gamma receptor (FcγR) profiles after overnight culture, showing the expression of CD16 and CD32% positive and MFI). (E) Expression of activating and cytotoxic markers after overnight culture: NKG2C, CD57, NKG2D, and DNAM-1% positive and MFI). In all bar graphs, red bars represent REST and blue bars represent EX. Bars show the mean ± SEM with paired donor values overlaid and connected by dotted lines. PBMCs from N = 10 healthy donors were used. Each condition was run in duplicate, and the results were averaged per donor. NK cells were defined as CD3−CD56+ lymphocytes. Statistical tests were two-sided. Panel A: paired t-test (EX vs. REST). Panel B: one-way repeated-measures ANOVA within each exercise state (REST or EX), followed by Dunnett’s multiple comparisons test vs. The control group. Panels C–E: two-way repeated-measures ANOVA with factors for exercise (REST vs. EX) and regimen (e.g., control vs. DRd), followed by Tukey’s multiple comparisons test.Significance is indicated by asterisks or exact p-values where appropriate. Thresholds were set at *p < 0.05, **p < 0.01, and ***p < 0.001. “ns” denotes not significant. Abbreviations: PBMC, peripheral blood mononuclear cell; NK, natural killer; MFI, mean fluorescence intensity; DRd, daratumumab/lenalidomide/dexamethasone; MRd, magrolimab/lenalidomide/dexamethasone

Combination therapies sensitize myeloma cells by downregulating inhibitory ligands

To determine whether MM therapies alter tumor susceptibility to NK cell killing, we examined ligand expression on myeloma cells after overnight treatment with DRd or MRd (Supplementary Fig. 1). Both regimens significantly reduced the percentage of HLA-E–expressing cells (p ≤ 0.0105), and DRd also lowered PD-L1 expression (p = 0.0425). MHC class I remained unchanged, whereas MHC class II MFI increased with both treatments (p ≤ 0.0454). No significant changes were observed in major activating ligands (MICA/B, ULBP family) or the FAS receptor. Thus, MM therapies sensitize tumor cells primarily by downregulating inhibitory checkpoints, while the exercise-driven synergy likely arises from immune cell rather than tumor cell modifications.

Exercise enhances anti-myeloma activity through increased NK cell numbers and improved per-cell function under therapeutic stress

To distinguish whether exercise-enhanced anti-myeloma activity (Fig. 2A–D) was driven by NK cell abundance or per-cell function, we compared cytotoxicity of purified NK-cells from rest and exercise conditions. In untreated controls, no significant differences were observed between groups. Similarly, under pre-treatment conditions (Fig. 4A), both daratumumab- and magrolimab-based therapies increased cytotoxicity (main drug effects: p = 0.005 and p = 0.003), but exercise had no independent effect per cell, with only a trend toward a drug × exercise interaction under DRd (p = 0.075). In contrast, co-treatment assays revealed significant drug × exercise interactions for both DRd (p = 0.0032) and MRd (p = 0.0250) (Fig. 4B). Post hoc testing showed that exercise-mobilized NK-cells displayed greater cytotoxicity than resting NK-cells with DRd (p = 0.0154), daratumumab alone (p = 0.0324), and magrolimab alone (p = 0.0138). Daratumumab alone with exercise yielded the highest cytotoxicity, exceeding DRd (p = 0.0461) and trending above control (p = 0.0511). A similar pattern emerged with magrolimab-based therapies: exercise-mobilized NK-cells outperformed resting NK-cells with magrolimab alone (p = 0.0138), though not with MRd (p = 0.4391). Comparisons among control, MRd, and magrolimab-alone groups confirmed that magrolimab alone significantly outperformed control under both rest (p = 0.0229) and exercise (p = 0.0074) conditions, with additional superiority over MRd under exercise (p = 0.0441). Together, these findings indicate that exercise enhances anti-myeloma effects largely by expanding NK cell numbers, while the surviving exercise-mobilized NK-cells also display superior per-cell cytotoxicity under therapeutic stress.

Fig. 4.

Exercise enhances purified NK cell cytotoxicity via a CD16-dependent mechanism. (A) Pre-treatment: target cells were exposed to the drug regimens before co-culture with purified natural killer (NK) cells. (B) Co-treatment: both target cells and purified NK cells were exposed to the drug regimen overnight before co-culture. (C) Pre-treatment: target cells were exposed to antibodies before being co-cultured with NK cells. (D) Co-treatment: both target cells and NK cells were exposed to antibodies overnight prior to co-culture. (A-B) The bar graphs show the percentage of specific lysis for control conditions, combination therapies DRd (daratumumab + lenalidomide + dexamethasone) and MRd (magrolimab + lenalidomide + dexamethasone), and their respective monoclonal antibodies alone, daratumumab (D) and magrolimab (M). (C-D) Assays were run with control (no antibody), daratumumab alone (D), and magrolimab alone (M). The effect of CD16 blockade was tested in the “D (anti-CD16)” and “M (anti-CD16)” groups.Red bars represent NK cells collected at rest (rest), while blue bars represent NK cells collected during acute exercise (exercise) at 80% VO2max. Data are presented as mean ± SEM. Individual dots represent data from each donor (N = 6), with lines connecting paired rest and exercise samples. Statistical comparisons shown are for the effect of exercise (exercise vs. Rest) within each treatment group. Significance is indicated as *p < 0.05; “ns” denotes not significant. The analysis was performed using a two-way repeated-measures ANOVA for pre- and co-treatment conditions separately (factors: exercise and regimen), followed by Tukey’s multiple comparisons test. Abbreviations: D, daratumumab; DRd, daratumumab/lenalidomide/dexamethasone; M, magrolimab; MRd, magrolimab/lenalidomide/dexamethasone

CD16 mediates the exercise-enhanced anti-myeloma effect

To determine whether CD16, the principal Fcγ receptor for NK cell ADCC, mediates the exercise effect, we performed CD16-blockade assays with purified NK-cells from rest and exercise conditions (Fig. 1B). NK-cells were pre-incubated with anti-CD16 before co-culture with MM1.S targets. Drug treatment had a significant overall effect on cytotoxicity (p < 0.001), but CD16 blockade abolished this effect: for both daratumumab and magrolimab, cytotoxicity fell to control levels when CD16 was blocked (Fig. 4C–D). In pre-treatment assays, exercise had no independent effect with daratumumab (p = 0.1280) and showed only a trend with magrolimab (p = 0.0661) (Fig. 4C). In co-treatment assays, significant drug × exercise interactions were detected (daratumumab p = 0.0032; magrolimab p = 0.0250), with exercise-enhanced cytotoxicity evident for both drugs (daratumumab p = 0.0476; magrolimab p = 0.0286) (Fig. 4D). However, these effects were dampened with CD16 blockade, with only nonsignificant trends remaining (daratumumab p = 0.0902; magrolimab p = 0.0867). These results identify CD16 engagement as a key mechanism underlying antibody-augmented cytotoxicity and the exercise-dependent enhancement of NK cell anti-myeloma activity.

Discussion

This study was designed to test whether exercise-mobilized lymphocytes could enhance the effectiveness of clinically relevant MM therapies under two modeled scenarios (1): a pre-treatment setting, in which lymphocytes collected during exercise were used as a potential donor lymphocyte infusion (DLI-X) following exposure of MM cells to standard regimens, and (2) a co-treatment setting, mimicking the potential impact of exercise performed during ongoing monoclonal antibody therapy. Acute exercise substantially increased the pool of circulating cytotoxic lymphocytes, particularly CD16⁺ NK-cells, which drove greater anti-myeloma activity when combined with daratumumab (anti-CD38) or magrolimab (anti-CD47). The enhancement was most consistent in the co-treatment model, where exercise-mobilized NK-cells exhibited superior ADCC compared with resting NK-cells. Notably, the benefits of combining exercise with daratumumab were attenuated by fratricide of CD38⁺ NK-cells, whereas magrolimab avoided this limitation, preserving the potent NK-cell populations mobilized during exercise. These findings highlight how the timing of exercise relative to therapeutic administration, as well as the choice of antibody target, may critically influence treatment outcomes.

Our results extend prior work by Collier-Bain and colleagues demonstrating that a single exercise bout can enhance ADCC against hematological cancers [26, 35]. Key gaps in this literature included whether such effects would persist in the presence of lenalidomide and dexamethasone—standard components of MM therapy—and whether they were driven primarily by increased NK-cell abundance or by intrinsic changes within the NK-cell compartment. We found that mixed lymphocytes collected after exercise were more effective at killing both dexamethasone-sensitive and resistant MM cell lines than their resting counterparts, even in the absence of treatment. Both DRd and MRd further enhanced cytotoxicity, but exercise-mobilized lymphocytes were consistently more responsive than resting cells, with the exception of the DRd co-treatment condition, where daratumumab-mediated fratricide of CD38⁺ NK-cells likely impaired activity.

To determine whether exercise also conferred intrinsic changes within NK-cells, we tested purified NK-cells. In the absence of therapy, resting and exercise-mobilized NK-cells were equally effective, confirming that the natural cytotoxicity observed in mixed lymphocyte populations was abundance-driven, consistent with our prior work [36]. However, purified exercise-mobilized NK-cells exhibited superior killing when treated with daratumumab- or magrolimab-containing MM therapies, indicating that exercise also enhances intrinsic ADCC capacity. Mechanistically, we confirmed that exercise preferentially mobilizes CD16⁺ (FcγRIIIA) NK-cells—key mediators of ADCC—and increases expression of the maturation marker CD57 as well as activating receptors NKG2D and DNAM-1. The primary role of CD16 was confirmed by blockade experiments, which abrogated the exercise-induced enhancement of ADCC. Nonetheless, residual enhanced activity was still observed, suggesting that the enrichment of a more activating and less inhibitory NK-cell phenotype also contributes. This is therapeutically significant, as standard MM therapies containing dexamethasone can suppress NK-cell function and receptor expressions. Therefore, exercise may counteract these immunosuppressive effects by selectively mobilizing a highly differentiated, mature CD16⁺ NK-cell subset with intrinsically greater ADCC capacity. Indeed, this is consistent with our prior work showing that while increased cytolysis against U266 and RPMI.8266 MM MM cell lines by exercise-mobilized lymphocytes were primarily abundance-driven, the per-cell killing capacity was enhanced 1 h into recovery and strongly correlated with NK-cell expression of CD158b, NKG2A, and NKG2C [36].

Consistent with prior work, we also found that exercise increased CD38 expression on NK-cells [26], rendering them more susceptible to daratumumab-induced fratricide and partially explaining the blunted efficacy of DRd co-treatment. In contrast, magrolimab—an anti-CD47 antibody with lower fratricide potential—did not show this limitation. Although CD47 was broadly expressed on NK-cells, its expression did not increase with exercise, unlike CD38. As a result, NK-cells mobilized during exercise retained their cytotoxic potential when combined with magrolimab, leading to greater overall activity in MRd compared with DRd. Phenotypic analysis supported this conclusion. The beneficial effects of exercise on the NK-cell phenotype were partially attenuated following exposure to both DRd and MRd. This finding is consistent with prior reports that lenalidomide and dexamethasone can suppress NK-cell function, in part by downregulating activating receptors such as NKG2D [37, 38]. Importantly, however, MRd better preserved the advantageous exercise-induced phenotype by maintaining higher expression of CD16 and DNAM-1, whereas DRd induced more detrimental changes compounded by NK-cell fratricide. Indeed, NK-cell frequencies among exercise-mobilized lymphocytes were significantly reduced after DRd exposure compared with both untreated and MRd conditions.

We also examined how DRd and MRd pretreatment altered tumor cell phenotype and susceptibility to exercise-mobilized NK-cells. Prior work by Thangaraj and colleagues [33, 39] showed that daratumumab-based triple therapies can strongly sensitize myeloma cells to NK-mediated killing by upregulating activating ligands (MICA/B, ULBPs, Fas) while downregulating inhibitory MHC class I molecules. In contrast, our findings were more modest. Neither DRd nor MRd induced significant upregulation of activating ligands on MM.1S cells. Instead, the dominant effect was a reduction in inhibitory ligands HLA-E and PD-L1. This suggests that the synergy observed in our study was not driven by dramatic tumor cell sensitization, but rather by the combined effects of exercise-induced mobilization of phenotypically primed NK-cells together with a modest alleviation of inhibitory checkpoint signaling on the tumor.

From a translational perspective, our findings offer distinct implications for both concurrent treatment regimens and adoptive immunotherapies. For patients undergoing active treatment, while exercise is a proven adjuvant that improves survival and tumor infiltration [9, 14, 15, 40], our results caution against integrating exercise into daratumumab-based regimens, where benefits may be compromised by exacerbated NK cell fratricide. To effectively harness exercise-induced enhancement in ADCC, our data indicate that non-fratricidal mAbs like magrolimab are required to preserve the mobilization of NK cells and enable synergy. On the other hand, our pre-treatment model supports an exercise-mobilized Donor Lymphocyte Infusion (DLI-X) strategy as a robust application for both treatment contexts. By harvesting effectors cells from donors during exercise and controlling the infusion timing, we could circumvent the potential fratricide associated with daratumumab while maintaining the enhanced cytotoxicity of the exercise-mobilized donor lymphocyte collection.

The distinction between CD38 and CD47 targeting is mechanistically critical to our findings. Daratumumab-induced fratricide occurs because activated NK cells—particularly the highly cytotoxic subsets mobilized by exercise—upregulate surface CD38, inadvertently becoming targets for the therapeutic antibody. This self-depletion significantly undermines the potential adjuvant benefit of exercise. In contrast, blocking the CD47-SIRPα axis enhances tumor phagocytosis and ADCC without compromising the effector pool. We acknowledge that the clinical development of magrolimab (an anti-CD47 mAb) has faced recent challenges, including the discontinuation of Phase III trials in AML and MDS due to futility or safety signals (NCT05079230; NCT05627466). However, the failure of specific magrolimab regimens in myeloid malignancies does not invalidate CD47 as a therapeutic target in Multiple Myeloma, nor does it negate the fundamental biological synergy observed here. Rather, our data support the broader investigation of CD47-SIRPα checkpoint inhibitors—or next-generation molecules with improved safety profiles—as the optimal partners for exercise-based immunotherapy, ensuring that the mobilized NK cells are preserved for tumor elimination.

Although these findings provide important mechanistic insight, several limitations warrant consideration. First, we acknowledge that potential confounding factors were present, including donor variabilities in training experience, fitness, and body composition. However, despite the heterogeneities, all participants demonstrated robust exercise-induced lymphocyte mobilization consistent with prior literature. As a mechanistic translational proof-of-concept, the study was not powered to examine between-subject modifiers, yet the observed effects were consistent across donors. Second, our experiments used an ex vivo system that cannot fully capture the complexity of the bone marrow microenvironment or systemic drug metabolism. Moreover, our reliance on immortalized multiple myeloma cell lines as targets may not fully recapitulate the biological heterogeneity and drug resistance patterns found in primary patient tumors. Similarly, lymphocytes were obtained from healthy donors, which may not reflect the functional capacity of immune cells from patients with MM, who are often older, immunosuppressed, and treatment-experienced [24]. Lastly, our study utilized acute drug exposures and did not model the effects of chronic dosing schedules or other key components of modern MM therapy, such as proteasome inhibitors or novel immunotherapies like BCMA-directed bispecific antibodies. Despite these limitations, the preclinical evidence presented here provides a compelling rationale for advancing this line of investigation. Future studies should include in vivo models to confirm the efficacy and safety of DLI-X in combination with standard MM treatment regimens and explore the impact of chronic exercise training on these responses. It will also be critical to determine whether the synergy we observed is sustained in the context of current quadruplet treatment regimens (e.g. D-VRd or Isa-VRd). Ultimately, pilot clinical trials combining structured exercise with non-fratricidal monoclonal antibody regimens may establish a new paradigm for enhancing immunotherapy in patients with MM.

In conclusion, an acute bout of exercise enhances anti-myeloma immunity by increasing NK-cell abundance and, in the presence of monoclonal antibodies, boosting NK-cell ADCC through enhanced CD16 expression. These benefits were consistent with anti-CD47 therapy but limited with anti-CD38 therapy due to NK-cell fratricide. Pairing exercise with non-fratricidal antibodies or leveraging exercise-mobilized donor lymphocyte infusions may represent promising strategies to optimize immunotherapy in multiple myeloma.

Electronic supplementary material

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Abbreviations

ACT

Adoptive Cell Transfer

ADCC

Antibody-Dependent Cellular Cytotoxicity

ANOVA

Analysis of Variance

ASCT

Autologous Stem Cell Transplantation

BMI

Body Mass Index

D

Daratumumab (when used alone)

DLI

Donor Lymphocyte Infusion

DLI-X

Exercise-Mobilized Donor Lymphocyte Infusion

DRd

Daratumumab, Lenalidomide, and Dexamethasone

D-VRd

Bortezomib, Daratumumab, Lenalidomide, Dexamethasone

E:T

Effector-to-Target

FcγR

Fc-gamma Receptor

GVHD

Graft-Versus-Host Disease

Isa-VRd

Isatuximab, Bortezomib, Lenalidomide, Dexamethasone

M

Magrolimab (when used alone)

MFI

Mean Fluorescence Intensity

MM

Multiple Myeloma

MRd

Magrolimab, Lenalidomide, and Dexamethasone

NK

Natural Killer

PBMC

Peripheral Blood Mononuclear Cell

PI

Propidium Iodide

SEM

Standard Error of the Mean

VO₂max

Maximal Oxygen Uptake

Author contributions

LC was responsible for the methodology, investigation, formal analysis, data curation, and writing the original draft. AMV and LMM contributed to the methodology, investigation, and data curation. FLB contributed to the conceptualization, methodology, investigation, formal analysis, and data curation. EK was responsible for conceptualization, funding acquisition, project administration, resources, visualization and supervision. RJS was responsible for conceptualization, funding acquisition, formal analysis, project administration, resources, software, supervision, validation, and visualization. All authors contributed to the review and editing of the manuscript and approved the final submitted version.

Funding

This work was supported by the National Institutes of Health (R01CA277493).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the University of Arizona Institutional Review Board (IRB Parent Protocol ID: 1801161041) and was conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent prior to their inclusion in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.