Dasatinib reversibly disrupts T cell motility and activation in lymph nodes

Daniela Claudino Carvoeiro; Ana Marcos-Jiménez; Ilya Tsukalov; Clara Viñas i Palou; Sergio Sanchez Luquin; Ihssane Eladnani; Raquel Hernanz; Ana M Briones; Enrique Martín-Gayo; Cecilia Muñoz-Calleja; Jens V Stein

doi:10.1126/sciadv.aec5951

. 2026 Aug 5;12(32):eaec5951. doi: 10.1126/sciadv.aec5951

Dasatinib reversibly disrupts T cell motility and activation in lymph nodes

Daniela Claudino Carvoeiro ^1,^†, Ana Marcos-Jiménez ^2,^3,^†, Ilya Tsukalov ^2,³, Clara Viñas i Palou ¹, Sergio Sanchez Luquin ¹, Ihssane Eladnani ¹, Raquel Hernanz ^4,^5,⁶, Ana M Briones ^4,^5,⁷, Enrique Martín-Gayo ^2,^8,⁹, Cecilia Muñoz-Calleja ^2,^3,^9,^*, Jens V Stein ^1,^*

^¹Department of Oncology, Microbiology, and Immunology, University of Fribourg, Fribourg, Switzerland.

^²Instituto de Investigación Sanitaria del Hospital Universitario La Princesa (IIS-Princesa), Madrid, Spain.

^³Departamento de Medicina, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain.

^⁴Hospital La Paz Institute for Health Research (IdiPaz), Madrid, Spain.

^⁵CIBER Cardiovascular (CIBERCV), Instituto de Salud Carlos III, Madrid, Spain.

^⁶Department of Basic Health Sciences, Universidad Rey Juan Carlos, Madrid, Spain.

^⁷Department of Pharmacology, Faculty of Medicine, Universidad Autónoma de Madrid, Madrid, Spain.

^⁸Centro de Biología Molecular Severo-Ochoa, CSIC-UAM, Madrid, Spain.

^⁹Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFC), Instituto de Salud Carlos III, Madrid, Spain.

Corresponding author. Email: cmunozc@salud.madrid.org (C.M.-C.);

Corresponding author. Email: jens.stein@unifr.ch (J.V.S.)

^†

These authors contributed equally to this work.

Roles

Daniela Claudino Carvoeiro: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing - review & editing

Ana Marcos-Jiménez: Conceptualization, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft

Ilya Tsukalov: Conceptualization, Formal analysis, Investigation, Methodology, Resources, Validation, Writing - original draft, Writing - review & editing

Clara Viñas i Palou: Formal analysis, Investigation, Methodology

Sergio Sanchez Luquin: Formal analysis, Investigation

Ihssane Eladnani: Formal analysis, Investigation, Validation

Raquel Hernanz: Conceptualization, Formal analysis, Investigation

Ana M Briones: Investigation, Visualization, Writing - review & editing

Enrique Martín-Gayo: Formal analysis, Investigation, Methodology, Resources, Supervision, Validation, Visualization, Writing - review & editing

Cecilia Muñoz-Calleja: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing - review & editing

Jens V Stein: Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Visualization, Writing - original draft, Writing - review & editing

PMCID: PMC13440384 PMID: 42555708

Abstract

The tyrosine kinase inhibitor dasatinib is prescribed as a first-line treatment for Philadelphia chromosome–positive leukemias and is increasingly used in other clinical settings. While dasatinib is effective and generally well tolerated, patients often experience a transient lymphocytosis attributable to a dasatinib-induced spleen contraction and an acute mobilization of splenocytes, as we have recently demonstrated in murine models and human patients. In contrast, the impact of dasatinib on lymphocyte surveillance of other secondary lymphoid organs, such as peripheral lymph nodes (LNs), has remained unclear. Here, we report that while dasatinib treatment selectively disrupted in vitro chemotaxis of B but not T cells, it caused a substantial, transient reduction of the in vivo interstitial motility in both subsets. The broad in vivo inhibition of T and B cell motility correlated with dasatinib-induced vasoconstriction of blood vessels and increased hypoxia within LNs. Pharmacological inhibition of the contraction-promoting factor Rho-associated coiled-coil kinase (ROCK) reversed dasatinib-induced LN hypoxia and rescued CD8⁺ T cell motility. Last, dasatinib treatment delayed but did not inhibit dendritic cell–mediated CD8⁺ T cell activation and proliferation within LNs. Overall, our findings uncover a dasatinib-induced transient disruption of the local adaptive immune system, which is distinct from the previously observed spleen contraction. From a clinical point of view, the disruption-recovery cycle of physiological immune surveillance induced by daily dasatinib treatment does not appear to cause a substantial impairment of key pathways underpinning CD8⁺ T cell activation.

The tyrosine kinase inhibitor dasatinib reversibly impairs T cell immune surveillance in lymph nodes.

INTRODUCTION

Chronic myeloid leukemia (CML) is characterized by the t (9;22) translocation, resulting in the Philadelphia chromosome (Ph⁺) and the expression of the oncogenic fusion protein BCR-ABL, which drives the uncontrolled proliferation of leukemic cells. Dasatinib, a dual inhibitor of BCR-ABL and Src tyrosine kinases, is a first-line treatment for Ph⁺ CML in both adults and children. It is also approved for Ph⁺ acute lymphoblastic leukemia (ALL) when combined with chemotherapy (1–3). In addition, dasatinib is used as maintenance therapy following hematopoietic cell transplantation in high-risk patients with Ph⁺ ALL (4). Its therapeutic versatility has expanded to include broader immunotherapeutic strategies; for example, it is now being combined with monoclonal antibodies (mAbs) such as blinatumomab (5–7). The inhibitory activity of dasatinib extends beyond BCR-ABL, targeting multiple off-target kinases such as PDGFR, EphA2, c-KIT, and those involved in cytoskeletal and chromatin remodeling, as well as in lymphocyte activation (8). Its broad spectrum of activity has motivated preclinical investigation as a treatment in solid tumors and T cell malignancies, including angioimmunoblastic T cell lymphoma and T cell ALL, which are linked to dysregulated T cell receptor (TCR) signaling (9–16). Furthermore, dasatinib enhances the efficacy of chimeric antigen receptor (CAR)–expressing T cells by preventing their exhaustion, which is triggered by sustained tonic signaling. In addition, its ability to alleviate severe side effects, such as cytokine release syndrome associated with CAR T cell and bispecific antibody therapies, has prompted clinical trials exploring its combination with CAR T cell immunotherapy for B cell malignancies (13, 14, 17–19). These developments illustrate the evolving role of dasatinib in the treatment of hematological malignancies. Hence, it is essential to elucidate the mechanism of action underlying its therapeutic as well as potential adverse effects.

One of the most intriguing side effects is a transient lymphocytosis, i.e., a temporary increase in circulating lymphocytes shortly after dasatinib administration (20). This phenomenon has been linked to improved treatment outcomes (21, 22), yet the underlying mechanism was only recently clarified. By integrating data from preclinical mouse models and human patients, we found that dasatinib induces a temporary contraction of the spleen stroma, resulting in the expulsion of splenocytes into circulation (23). While this finding underscored the physiological impact of dasatinib on the systemic adaptive immune system, local effects on lymphocyte trafficking and physiology within other secondary lymphoid organs (SLOs) remain largely unexplored. Peripheral lymph nodes (LNs)—essential components of the SLO network—are crucial for regional adaptive immunity, as they facilitate T cell surveillance and orchestrate interactions with antigen-presenting dendritic cells (DCs). To date, the impact of dasatinib on LN function and T cell behavior within this vital microenvironment has not been characterized.

In this study, we used preclinical mouse models to investigate the effects of dasatinib treatment on LNs. While dasatinib does not induce LN contraction as observed in spleen (23), intravital imaging revealed a potent but transient disruption of the interstitial motility in T and B cells, whereas it only affected B cell motility in vitro. This broad reduction in interstitial motility gradually returned to baseline levels after 24 hours in line with dasatinib pharmacokinetics and correlated with a vasoconstrictory response shortly after dasatinib administration. Reduced blood supply was reflected by increased lymphoid tissue hypoxia, which was reversed by pharmacological inhibition of the contractility-promoting Rho-associated coiled-coil kinase (ROCK) pathway. When examining the impact of dasatinib on CD8⁺ T cell responses, we found that only continuous exposure to peak dasatinib levels transiently achieved after uptake, but not lower concentrations, impaired mouse and human CD8⁺ T cell activation in vitro. Similarly, a transient hypoxia did not reduce early in vitro CD8⁺ T cell activation. Accordingly, the transient disruption of immunosurveillance by dasatinib delayed but did not inhibit DC-mediated CD8⁺ T cell activation and proliferation. Our findings provide evidence that dasatinib-induced alterations in LN physiology transiently impair lymphocyte dynamics without severely disrupting the development of a cellular immune response, offering insights into its immunomodulatory effects and their potential implications for adaptive immune function.

RESULTS

Dasatinib impairs B but not T cell in vitro chemotaxis

Homeostatic chemokines including the CCR7 ligands CCL19 and CCL21, the CXCR4 ligand CXCL12, and the CXCR5 ligand CXCL13 mediate trafficking of T and B cells into and within their lymphoid microenvironments (24–27). We performed Transwell migration assays with naïve murine T and B cells to assess the impact of dasatinib on chemokine-induced motility in a reductionist system. Dasatinib treatment significantly reduced B cell migration toward CCL21 (Fig. 1A), CXCL12 (Fig. 1B), and CXCL13 (Fig. 1C) across both 5- and 3-μm pore sizes (Fig. 1A), even at dasatinib concentrations as low as 10 nM. In contrast, dasatinib only induced a mild reduction on CD4⁺ T cell chemotaxis toward CXCL12 but not CCL21, while CD8⁺ T cell chemotaxis remained unaffected (Fig. 1, A and B). These findings align with data using human lymphocytes, where T cell migration to CXCR4 and CCR7 ligands remained intact under dasatinib treatment (28). At the concentrations used here, dasatinib did not affect chemokine receptor levels or cell viability (23), suggesting a direct effect on factors involved in chemokine receptor signaling pathways such as Btk, a tyrosine kinase expressed in B cells (29).

Fig. 1. — (A to C) Transwell chemotaxis of CD4⁺, CD8⁺, and B220⁺ cells to CCL21 across 5- and 3-μm pore sizes (A), and to CXCL12 (B) and CXCL13 (C) with 5-μm pore sizes. (D) Experimental layout of under agarose confinement assay. (E) Representative T and B cell tracks (with CCL21 and CXCL13, respectively, and ICAM-1) in under agarose assays. Scale bar, 20 μm. (F) T and B cell speeds and meandering index with or without dasatinib in under agarose confinement assays (n = 580 to 1351 tracks per condition). Data in (A) to (F) were pooled from at least two experiments and analyzed using analysis of variance (ANOVA) with Dunnett’s test against control [“DMSO”; (A) to (C)] and a Mann-Whitney test (F). *P < 0.05; **P < 0.01; ***P < 0.001.

To recreate a more physiologically relevant model, we performed under agarose confinement assays on chemokine and intercellular adhesion molecule–1 (ICAM-1)–coated surface. This assay mimics key aspects of lymphocyte motility observed in lymphoid tissue, such as confinement and track speeds (Fig. 1D) (30, 31). In this assay, T cells migrate faster and more straight as compared to B cell tracks, reflecting in vivo observations (Fig. 1E) (32). Addition of dasatinib in this setting did not reduce T cell speeds, with only a minor effect on cell directionality (assessed by the meandering index), whereas B cell speeds and directionality were consistently reduced by dasatinib (Fig. 1F). In summary, dasatinib exerts a B cell–restricted inhibitory effect on their ex vivo migratory capacity triggered by homeostatic chemokine signaling.

Dasatinib induces a profound yet transient decrease in interstitial T and B cell motility

To examine whether our ex vivo results translate into impaired lymphocyte motility in vivo, we studied the behavior of adoptively transferred, fluorescently labeled naïve T and B cells as live probes for immune cell surveillance and treated C57BL/6 mice with dimethyl sulfoxide (DMSO) or dasatinib (50 mg/kg) prior to intravital two-photon microscopy (2PM) of surgically prepared popliteal LNs (Fig. 2A). We then performed an offline analysis of key dynamic track parameters including cell speeds, meandering index, arrest coefficient (fraction of track segments with speeds below 4 μm/min), and the track displacement (Euclidian distance of track start and end points; Fig. 2B). Unexpectedly, our findings revealed that dasatinib administration caused a strong and acute disruption of both interstitial T cell and B cell motility at 2 hours postadministration, which affected all the analyzed parameters (Fig. 2, C and D, and movie S1). Additional experiments confirmed a significant decrease of T cell and B cell speeds at a 10-fold lower dasatinib dose (fig. S1).

Fig. 2. — (A and B) Experimental layout (A) and evaluated track data parameters (B). Scale bars, 30 μm. Images in (B) depict the same field of view (FOV) showing a single time point (left) and the overlaid tracks of all time points (right). (C) Representative T cell (green) and B cell (red) tracks at 2 hours after DMSO and 2, 8, or 24 hours after dasatinib treatment (50 mg/kg). Scale bar, 30 μm. (D) Speeds, meandering index, arrest coefficient, and track displacement of T cells and B cells at 2 hours after DMSO and 2, 8, or 24 hours after dasatinib treatment (n = 2 mice per group from two independent experiments with 89 to 1056 tracks analyzed per condition). Data in (D) were analyzed using ANOVA with Dunnett’s test against control (“DMSO”). ***P < 0.001. h, hours. HEV, high endothilial values; p.i., postinfection.

Based on the rapid clearance of dasatinib from the blood stream (20, 33), we assessed whether motility parameters recovered following the initial disruption of lymphocyte motility. In line with the transient nature of dasatinib-induced spleen contraction, T and B cell motility parameters improved 8 hours after the initial treatment and had almost completely normalized after 24 hours (Fig. 2, C and D, and movie S1). Together, our data show that dasatinib strongly affects both key T and B cell motility parameters in vivo shortly after drug uptake, in contrast to its restricted impact on B cell chemotaxis in vitro.

We examined whether the impact of dasatinib was also observed in memory T cells in lymphoid and nonlymphoid tissues. To this end, we generated chimeric mice harboring green fluorescent protein–positive (GFP⁺) OT-I memory CD8⁺ T cells (T_MEM) after a systemic viral infection and examined the motility parameters of central memory T cells (T_CM) in LNs and tissue-resident memory T cells (T_RM) in submandibular salivary glands (SMGs) before and after dasatinib treatment (fig. S2A). In both organs, dasatinib treatment induced a rapid decrease in T_CM and T_RM speeds and directionality (fig. S2, B and C), suggesting that dasatinib also affects both lymphoid and nonlymphoid organ surveillance across various T_MEM populations.

Dasatinib induces an increase in hypoxia in peripheral LNs but not spleen

We next examined the mechanism underlying dasatinib-induced disruption of lymphocyte surveillance, focusing on the dynamic regulation of blood irrigation in LNs (34). Recent work by the Mueller group has uncovered that noradrenaline (NA) administration induces a rapid loss of intranodal T and B cell motility by inducing vasoconstriction, which reduces LN blood supply and causes hypoxia (35). Since oxygen supply is critical to maintain intranodal migration of naïve T cells (36) and as dasatinib induces contraction of human endothelial cells in a ROCK-dependent manner (37), we sought to determine whether dasatinib exerts an effect on vasoconstriction in LNs. In control experiments, we used intravital 2PM to study blood vessel diameters in the T cell zone of popliteal LNs (Fig. 3A), where we confirmed the vasoconstrictory effect following NA administration (Fig. 3, B and C). Akin to NA, dasatinib administration induced a consistent constriction of the LN vasculature at 2 hours postadministration (Fig. 3, B and C). To examine whether vasoconstriction and reduced blood supply affected LN oxygenation, we administered the hypoxia probe pimonidazole to control and dasatinib-treated mice, followed by flow cytometry analysis (Fig. 3D). Dasatinib treatment induced increased pimonidazole levels as compared to control treatment, indicating cellular hypoxia in T cells, B cells, and macrophages in LNs but not spleens (Fig. 3E). To establish a link between dasatinib-induced hypoxia and vasoconstriction, we pharmacologically inhibited ROCK using the drug Y27632 and measured pimonidazole levels. Dasatinib-induced hypoxia was reversed when mice were cotreated with dasatinib and Y27632 (Fig. 3, F and G).

Hypoxia leads to an increase in intracellular Ca²⁺ levels (38, 39). Accordingly, NA induced a rapid and widespread wave of Ca²⁺ signaling in LN leukocytes (35). This suggests that Ca²⁺ signaling, triggered by oxygen-sensing mechanisms, may act as a regulatory pathway for controlling lymphocyte motility in response to hypoxic conditions. To investigate dasatinib-induced Ca²⁺ signaling in LNs, we adoptively transferred CMTMR-labeled T cells loaded with the Ca²⁺ indicator Calbryte 520 AM, a fluorescent indicator designed for the measurement of intracellular Ca²⁺ levels (Fig. 3H). Administration of dasatinib induced increased Ca²⁺ signaling in T cells coinciding with loss of T cell motility (Fig. 3, I and J).

Next, we performed 2PM imaging of LN after coadministration of dasatinib and Y27632 (Fig. 3K). Under this condition, vessel diameters remained unchanged (Fig. 3L), and T cell speeds and directionality were comparable to pretreatment conditions (Fig. 3M). In summary, our data align with a model in which dasatinib treatment leads to vasoconstriction and hypoxia in LNs, which is reversed by blocking ROCK activity.

In vitro dasatinib administration blocks DC-mediated CD8⁺ T cell activation at peak concentrations

We next focused on the impact of dasatinib treatment on CD8⁺ T cell activation, a lymphocyte subset with high relevance for antitumoral activity. Dasatinib has been shown to exert a direct inhibition on TCR-mediated signal transduction, cellular proliferation, and cytokine production, possibly by inhibiting Lck, a key kinase in the TCR signaling pathway (40, 41). Noting that in vivo serum concentrations of dasatinib in human patients transiently reach peak concentrations of ∼100 nM shortly after administration before decreasing over time (23), we supplied dasatinib at concentrations of 1, 10, and 100 nM in an in vitro DC-triggered murine CD8⁺ T cell activation model (Fig. 4A). In brief, we cocultured lipopolysaccharide (LPS)–activated DCs pulsed with the ovalbumin (OVA)_257–264 peptide, which binds the OT-I TCR in the context of H2K^b (42), together with CellTrace violet (CTV)-labeled OT-I T cells to track cell proliferation. After 48 hours, we investigated whether the continuous exposure to dasatinib, at clinically relevant concentrations, during CD8⁺ T cell activation directly affects cell proliferation and the expression of early activation markers. At none of these concentrations tested did we observe a significant decrease in the T cell proliferation index, although there was a trend toward reduced proliferation at 100 nM dasatinib (Fig. 4, B and C). Similarly, assessing CD69, CD25, and CD44 as indicators of T cell activation 48 hours poststimulation showed no effect of continuous dasatinib exposure to 1 and 10 nM, representing postpeak concentrations of dasatinib (Fig. 4, D to F). In contrast, continuous exposure of DC–OT-I T cell cocultures to 100 nM dasatinib significantly reduced changes in activation marker expression (Fig. 4, D to F). These data suggested that shortly after dasatinib uptake correlating with peak drug levels, a direct inhibitory effect on CD8⁺ T cell activation is likely to occur. However, under physiological conditions, this direct inhibitory effect is likely to wane quickly owing to the rapid clearance of dasatinib from the circulation.

Fig. 4. — (A) Experimental layout of peptide-pulsed DC and OT-I T cell in vitro activation. (B) Flow cytometry plots depicting CTV dilution on OT-I cells at 48 hours after transfer followed by DMSO or dasatinib treatment. (C) Proliferation index after DMSO or dasatinib treatment. (D to F) Flow cytometry plots depicting CTV dilution and CD69 (D), CD25 (E) and CD44 (F) expression and quantification of MFI on OT-I cells at 48 hours after DMSO or dasatinib in vitro treatment. One of two independent experiments (n = 2 mice per condition) with three replicates is shown. Data in (C) to (F) were analyzed using ANOVA with Dunnett’s test against “DMSO.” *P < 0.05; **P < 0.01; ***P < 0.001.

Transient hypoxia does not inhibit early CD8⁺ T cell activation

Given that hypoxia is a readily measurable result of dasatinib-induced reduction of blood supply, we examined whether this process might affect CD8⁺ T cell activation. To correlate data gained in mouse models with a clinically relevant setting, we took advantage of a human CD8⁺ T cell activation protocol by coculturing them with autologous human monocyte-derived dendritic cells (MDDCs) loaded with a cytomegalovirus (CMV) peptide pool (Fig. 5A). As control, we confirmed the inhibitory effect of continuous dasatinib exposure at 100 nM on CMV-specific CD8⁺ T cell activation, which resulted in reduced granule release (CD107a) and interferon-γ (IFNγ) production (Fig. 5, B to D). In contrast, a short (6 hours) exposure to hypoxic conditions did not reduce the ability of human CD8⁺ T cells to increase these markers (Fig. 5, C and D). Together, these data suggest that a transient decrease of oxygen levels, as in dasatinib-treated LNs, has no detrimental effect on initial CD8⁺ T cell activation.

Fig. 5. — (A) Experimental layout. (B) Representative flow cytometry plots gated on live human CD8⁺ T cells. (C and D) CD107 (C) and IFNγ (D) levels under normoxic and hypoxic conditions. Data are pooled from five independent experiments and analyzed using a paired Student’s t test. *P < 0.05.

Dasatinib administration delays but does not block DC-mediated CD8⁺ T cell activation

Based on in vitro activation data obtained using mouse and human CD8⁺ T cells (Figs. 4 and 5), we predicted that daily injections of dasatinib will not completely block in vivo CD8⁺ T cell activation. To address this point experimentally, we used a vaccination model of transferring LPS-activated DCs pulsed with the OVA_257–264 peptide into the foot hock of C57BL/6 mice. Eighteen hours later, we adoptively transferred CTV-labeled OT-I TCR transgenic T cells to track cell proliferation and activation parameters. To synchronize the initiation of T cell–DC interactions, we injected an anti-l-selectin antibody at 4 hours post–T cell transfer, which blocks further homing of circulating T cells into LNs (43). Dasatinib was administered at the time of T cell transfer and again at 24 hours, followed by flow cytometry at 48 hours post–T cell transfer (Fig. 6A). Dasatinib treatment resulted in reduced proliferation of OT-I cells in the reactive LNs (Fig. 6, B and C), thereby impairing T cell expansion (Fig. 6D). Flow cytometry further revealed a delay in the down-regulation of the early activation marker CD69 and concomitant delays in CD25 and CD44 induction in non- and early-divided OT-I T cells from dasatinib-treated recipients (Fig. 6, E to G). Nonetheless, these early CD8⁺ T cell activation parameters aligned with control-treated OT-I T cells during later cell divisions (≥4), indicating that dasatinib treatment delays but does not block T cell activation (Fig. 6, E to G). Last, we assessed effects on the late effector phase. Mice were DC-vaccinated as in Fig. 6A, given daily dasatinib, and analyzed 7 days posttransfer for frequency and phenotype of OT-I T cells. OT-I frequencies among CD8⁺ T cells were comparable regardless of dasatinib treatment (Fig. 6H). Analysis of CD127⁺KLGR1⁻ memory precursor effector cells (MPECs) versus CD127⁻KLGR1⁺ short-lived effector cells (SLECs) within the transferred OT-I population revealed a small shift toward SLECs in the spleen following dasatinib treatment (Fig. 6, I and J).

Fig. 6. — (A) Experimental layout of peptide-pulsed DC and OT-I T cell transfer. (B) Flow cytometry plots depicting CTV dilution on OT-I cells at 48 hours after transfer followed by DMSO or dasatinib treatment. (C and D) Proliferation index (C) and total OT-I T cell numbers (D) after DMSO or dasatinib treatment. (E to G) Flow cytometry plots depicting CTV dilution and CD69 (E), CD25 (F), and CD44 (G) expression and quantification of MFI on OT-I cells at 48 hours after transfer followed by DMSO or dasatinib treatment (n = 8 to 10 mice pooled from two independent experiments). (H) Percent OT-I of CD8⁺ T cells in spleen at day 7 post–DC vaccination. (I) Representative flow cytometry plots of CD127 and KLRG1 expression on CD44^high OT-I T cells at day 7 post–DC vaccination. (J) Quantification of MPEC and SLEC OT-I T cell frequencies at day 7 post–DC vaccination (n = 5 to 6 mice pooled from two independent experiments). Data in (C) to (G) and (I) were analyzed using unpaired Student’s t tests. *P < 0.05; ***P < 0.001.

In conclusion, our findings suggest that shortly after uptake, dasatinib disrupts adaptive immune surveillance in peripheral LNs by delaying T cell activation and impeding their rapid expansion. Nonetheless, a transient disturbance of LN physiology does not block the development of a cellular immune response (fig. S4).

DISCUSSION

The expanding applications of dasatinib in treating hematological malignancies call for a comprehensive analysis of its effects on the endogenous immune system. Our preclinical studies using mouse models reveal that dasatinib directly inhibited B cell chemotaxis in vitro and transiently disrupted B and T cell scanning in vivo. This disruption appears to be primarily driven by dasatinib-induced alterations of the lymphoid microenvironment, specifically vasoconstriction and hypoxia resulting from reduced blood flow. Focusing on CD8⁺ T cell responses, we observed that only continuous exposure to peak plasma concentrations of dasatinib, reached shortly after drug intake, significantly reduced CD8⁺ T cell proliferation. In contrast, early in vivo expansion and differentiation into short-lived effector and memory precursor populations were not strongly affected. In accordance, a transient exposure to low O₂ levels did not impair initial human T cell activation. Consequently, a single daily dose of dasatinib delays but does ultimately not block local CD8⁺ T cell activation and proliferation in LNs, suggesting that cellular immunity should be largely preserved in dasatinib-treated patients.

T and B cells use ligands for CCR7 and CXCR5, respectively, as guidance cues for their localization within the lymphoid microenvironment. In addition, these chemokines facilitate homeostatic scanning of antigen-presenting cells (30, 44–49). Although chemokine receptors on T and B cells share overlapping intracellular signaling pathways (50), notable differences exist. Most notably, phosphoinositide-3-kinase–dependent signaling pathways and their downstream effectors, such as Tec family kinases, are differentially linked in T and B cells (51–54). For example, CXCR5 mediates B cell chemotaxis primarily through the engagement of the B cell–specific Tec kinase Btk (29, 55), which is a known target of dasatinib (56). Furthermore, given the broad range of off-target effects of dasatinib, it may also inhibit additional, yet uncharacterized factors involved in B cell motility. This distinct molecular wiring likely accounts for the differential effects of dasatinib on B cell chemotaxis compared to T cells, even when both cell types migrate toward the same chemokines, such as the ligands for CXCR4 or CCR7.

Unexpectedly, we observed a significant decline in both T and B cell motility parameters between 2 to 8 hours following in vivo administration of dasatinib, followed by a recovery by 24 hours. This transient loss of motility correlated with dasatinib-induced vasoconstriction mediated via the ROCK pathway, indicating that the drug’s pharmacokinetics largely influence its effects on immune cell dynamics. Vasoconstriction and reduced blood flow result in hypoxia within LNs, which has been associated with decreased lymphocyte motility (35, 36). Under hypoxic conditions, elevated intracellular Ca²⁺ levels in T cells disrupt cytoskeletal organization and actomyosin-driven motility (57), a critical process for efficient immune surveillance. Of note, the transient decrease of lymphocyte motility is distinct from the persistent lack of immune cell surveillance. As example, the loss of immune cell migration in patients with a primary immunodeficiency caused by decreased expression of the promigratory signaling molecule DOCK2 (58) results in a severe combined immunodeficiency and susceptibility to respiratory infections (59, 60).

When assessing the physiological impact of dasatinib on cellular immunity, our in vivo DC vaccination model indicates that dasatinib causes a delay, but not complete suppression, of the CD8⁺ T cell response. While our in vitro experiments confirmed that dasatinib can inhibit CD8⁺ T cell activation (40, 41), this effect was only observed during continuous exposure to 100 nM dasatinib. Since this concentration is within the range of peak levels transiently reached following daily in vivo administration in patients (20, 23), it suggests that physiological dasatinib concentrations are not maintained at sufficiently high levels to exert a substantial inhibitory effect on CD8⁺ T cell activation in vivo. Similarly, we found that hypoxia did not affect early activation parameters in human CD8⁺ T cells. Hypoxia is a hallmark of inflamed, infected, or damaged tissues, occurring when the oxygen supply from the vasculature fails to meet cellular demands (61). This imbalance disrupts cellular metabolism, forcing a shift from efficient oxidative phosphorylation to glycolysis, which produces significantly less ATP and creates an environment unfavorable for optimal cell function. Furthermore, beyond hypoxia, reduced blood supply might affect metabolite exchange between blood and lymphoid tissue or indirectly delay T cell activation through stromal and hematopoietic cell interactions. Nonetheless, previous studies have shown that while CD8⁺ T cells cultured under low oxygen conditions proliferate more slowly than those in normoxia, they also develop enhanced cytotoxic activity (62). Similarly, for CAR T cell generation, 24 hours of hypoxia during initial priming suppresses T cell activation but induces a lasting shift in T cell differentiation, metabolism, and epigenetic markers, ultimately resulting in improved antitumor cytotoxicity in a mouse tumor model (63). Together, these data are in line with our observation that transient dasatinib-induced hypoxia does not exert a strong inhibitory effect on in vivo CD8⁺ T cell activation, a finding corroborated by studies using activated human T cells in vitro.

In a previous study, we identified a link between dasatinib-induced splenic stromal contractility and transient lymphocytosis (23). Our current data extend this understanding to LNs, where dasatinib influences immune cell behavior through vasoconstriction. These results provide important insights into the implications of using dasatinib as an immunotherapeutic adjunct and its effects on the patient’s endogenous immune system. Although dasatinib is known to modulate immune responses, such as affecting the persistence and function of CAR T cells (64, 65), our data suggest that cycles of disruption and subsequent recovery of physiological immune surveillance levels under daily dasatinib treatment do not impair robust CD8⁺ T cell activation. Thus, the rapid turnover of dasatinib limits unwanted side effects on the endogenous immune system.

As a limitation, our study does not evaluate T cell function at the memory phase of immune responses. In addition, while the link between blood supply, tissue oxygen levels, and T cell motility is well established (35, 36), vasoconstriction might also limit tissue exchange of metabolites and catabolites affecting T cell motility. A further limitation of our study is the impact of dasatinib on humoral responses. In the hen egg lysozyme-ovalbumin (HEL-OVA) immunization model, the initial antibody response was not impaired (fig. S3). Our findings are consistent with previous reports in tyrosine kinase inhibitor–treated patients, where T and B cell responses after vaccination against influenza and severe acute respiratory syndrome coronavirus 2 are preserved (66, 67). Furthermore, there is limited evidence of dasatinib-induced hypogammaglobulinemia (68), and dasatinib-treated patients do not require specific vaccination recommendations. Together, these results help explain why dasatinib does not induce long-term immunosuppression, while still showing utility in modulating T cell responses in clinically relevant therapeutic settings, such as the control of cytokine release syndrome.

MATERIALS AND METHODS

Mice

Six to 20-weeks-old male and female C57BL/6J mice (Janvier, AD Horst) or Tg(TcraTcrb)1100Mjb (“OT-I”) TCR transgenic mice backcrossed to GFP or tdTomato-expressing Ai14 × ZP3 (“tdTom⁺”) (42, 69–71) were used in this study. All animals were maintained at the University of Fribourg (Switzerland). All animal work was approved by the Cantonal Committees for Animal Experimentation and conducted in accordance with federal guidelines (License 2021-25-FR).

Cells isolation and culture, and general reagents for in vitro and in vivo use

For lymphocytes transfers or in vitro experiments, LNs and spleens were harvested and passed through cell strainers (70 μm; Bioswisstec). Cells were kept in complete medium (CM), which contains RPMI-1640 (Gibco, Life Technologies) supplemented with penicillin/streptomycin/glutamine (Gibco, Life Technologies), 10 mM Hepes (Gibco, Life Technologies), and 10% fetal bovine serum (FBS; Sigma-Aldrich). In some experiments, T and B cells were negatively isolated using Easy Sep T or B Cell Isolation Kits (STEMCELL Technologies). For 2PM experiments, after staining with Cell Trackers CMTMR, CFSE, or CTV (Thermo Fisher Scientific) according to manufacturer’s instructions, 2 to 5 × 10⁶ cells were resuspended in 100 μl of CM and intravenously transferred into recipient mice. Cell trackers were swapped between experiments. For calcium influx assay, cells were stained with 2 μM Calbryte 520 AM for 1 hour. Dasatinib (LC Laboratories) was reconstituted in DMSO and further diluted in CM. For in vivo experiments, intraperitoneal injections were performed. The anti-CD62L mAb (clone MEL14) was purchased from BioXcell, and 100 μg per mouse was injected intraperitoneally for blocking of homing into LNs. The ROCK inhibitor Y27632 (from Sigma-Aldrich, Hello Bio, or Selleckchem) was used at 10 mg/kg for intraperitoneal administration.

DCs were generated from C57BL/6 bone marrow by culturing for 7 days in CM containing 1 mM sodium pyruvate (P04-43100) and minimum essential medium essential amino acids (P08-32100, from PAN Biotech) supplemented with 10% culture supernatant of SP2/0 cells expressing Flt3-ligand, followed by activation with Salmonella typhimurium–derived LPS (0.5 μg/ml; Sigma-Aldrich) for 24 hours. Activated DCs used for vaccination were pulsed with 100 nM OVA_257–264 (EMC microcollections) for 45 min. For in vivo activation experiments, we intravenously transferred 5 × 10⁴ (for analysis on day 7) or 3 × 10⁶ T cells (for analysis on day 2) per mouse 18 hours after subcutaneous DC vaccination (1 × 10⁶ DCs in 10 μl per foot hock).

In vitro chemotaxis

Lymphocytes were allowed to migrate to chemokines as indicated in the figure legends using Transwell chambers (Costar). In brief, 0.5 × 10⁶ cells per well in CM were added to the top chambers of 3- or 5-μm pore size well and allowed to migrate for 2 hours before cell counting and analysis of CD4⁺, CD8⁺, and B cell ratios using flow cytometry.

Flow cytometry

Acquisition of the samples was performed using an LSR Fortessa (BD) cytometry system, and data were analyzed using FlowJo. The mAbs used for mouse samples were from BioLegend: CD4-PE/Cy7 (RM4-4), CD8-APC/Fire 750 (53-6.7), F4/80-BV421 (BM8), B220-AF647 (RA3-6B2), CD45.2-APC/Fire 750 (104), CD3ε-biotin (145-2C11), CD44-BV605 (IM7), CD69-BV711 (H1.2F3), CD25-APC (PC61), KLRG1-Pe-Cy7 (2F1), and CD127-PE (A7R34). Biotinylated mAbs were detected with streptavidin-BV711 (BioLegend). Zombie Red or Green (R&D) was used to label dead cells. The gating strategy started by selecting cells based on forward scatter (FSC) and side scatter (SSC), and singlets by forward scatter area (FSC-A) versus forward scatter height (FSC-H) parameters. Next, leukocytes were selected by a CD45⁺ gate. Then, CD4, CD8 (or CD3ε), B220, and F4/80 mAbs were used within lymphocytes to identify CD4⁺ T cells, CD8⁺ T cells (or T lymphocytes), B cells, and macrophages, respectively.

For human samples, acquisition was performed on a BD FACS Canto II. The mAbs used were anti-IFNγ–fluorescein isothiocyanate (FITC; BioLegend, 4S.B3), CD107a-APC (BioLegend, H4A3), CD8-APC/Cy7 (BioLegend, HIT8a), CD3-Pacific Blue (Immunostep, 33-2A3), and the Viability-Invitrogen LIVE/DEAD Fixable Yellow Dead Cell Stain. Cells were stained using the Fix&Perm kit (Invitrogen) and selected based on FSC versus SSC, followed by singlet selection with FSC-A versus FSC-H parameters. CD3 and CD8 were used to identify CD8⁺ T cells, and their expression of IFNγ and CD107a was analyzed.

Under agarose assays

A circle of 17-mm diameter was cut in the center of 60-mm dishes, and the hole was sealed using a 24-mm glass coverslip and aquarium silicone (Marina). After drying, we overlaid a 5-mm high ring cut from a 15-ml tube and sealed it with paraffin. Coverslips were coated with Protein A (20 μg/ml; BioVision, 6500-10) for 1 hour at 37°C, washed three times with phosphate-buffered saline (PBS), and blocked with 1.5% bovine serum albumin (BSA) for 1 hour at 37°C. Cover glasses were coated with 100 nM recombinant ICAM-1 Fc (R&D, 796IC) for 2 hours at 37°C before being washed twice with PBS. Coverslips were blocked with 1.5% BSA for 1 hour at 37°C before adding 500 nM CCL21 (Peprotech) or CXCL13 (R&D) in cell culture medium. For agarose overlay, 10 ml of 2× RPMI containing 20% FCS and 5 ml of 2× Hanks’ balanced salt solution were heated up to 56°C in the water bath. SeaKem Gold agarose (100 mg; Lonza, 50152) was heated in 5 ml of MilliQ water and added to the prewarmed medium to obtain a 0.5% agarose mix. After cooling down to 37°C, the chemokine solution was removed, and 500 μl of the agarose was added on the coverslip. The agar was left to solidify for 5 min at room temperature and subsequently for 20 min at 4°C. T cells or B cells were gently injected under the agarose in a volume of ∼2 to 5 μl directly prior to imaging. Time-lapse images were taken with a DeltaVision Elite wide-field fluorescence microscope equipped with a 20×, numerical aperture 0.75 objective (Olympus). Images were taken from 8 to 10 FOV every 20 s for 60 min. Data were analyzed using Imaris (Bitplane).

Intravital two-photon microscopy

C57BL/6 mice were anesthetized with ketamine/xylazine/acepromazine, and the right popliteal LN was surgically exposed without damaging the organ capsule or lymph vessels as described (72). Prior to imaging, AF633-conjugated MECA-79 (10 μg per mouse) or TRITC-dextran (500 kDa, Thermo Fisher Scientific) was injected intravenously to visualize high endothelial venules and the blood vasculature, respectively. Intravital imaging was performed with an Olympus BX50WI fluorescence microscope equipped with a 25× objective (Nikon, numerical aperture 1.1) and a TrimScope 2PM system controlled by ImSpector software (LaVision Biotec). Time-lapse image series were acquired between 50 and 150 μm below the LN surface using an automated tissue-drift correcting software (73). A Ti:sapphire laser (Mai Tai HP) was tuned to 780 or 840 nm. Alternatively, we used a Bergamo II multiphoton microscope system (Thorlabs) equipped with a Cronus 2P laser (Light Conversion). Image stacks were transformed into volume-rendered four-dimensional videos with Imaris (Bitplane), which was also used for semiautomated tracking of cell motility. In Ca²⁺ flux experiments using Calbryte 520, mean green fluorescence intensity was quantified using “surface” automated tracking in Imaris and normalized to mean CMTMR intensity. The diameter of blood vessels in LNs were measured using VasoMetrics, a macro written in ImageJ/Fiji for spatiotemporal analysis of microvascular diameter (74).

Infection model

Purified GFP⁺ OT-I CD8⁺ T cells (5 × 10⁴) were adoptively transferred into C57BL/6 recipient mice 1 day prior to infection with OVA-expressing lymphocytic choriomeningitis virus (LCMV-OVA; 1 × 10⁵ plaque-forming units intraperitoneally) (75). At >28 days postinfection, SMGs and popliteal LNs were surgically exposed for 2PM, and memory CD8⁺ T cell speeds and meandering index were examined before and 1 hour post–dasatinib treatment (50 mg/kg) (76).

Assessment of in vivo hypoxia using pimonidazole staining

The Hypoxyprobe Plus Kit (HP2-200kit, Hypoxyprobe) was used as per the manufacturer’s instructions. Briefly, mice received pimonidazole hydrochloride (30 mg/kg) intraperitoneally immediately after dasatinib injection. At the indicated time points, mice were sacrificed; brachial, inguinal, axillary, and popliteal LNs and spleens were harvested; and the cell suspension was prepared. For pimonidazole staining, cells were fixed with the Cytofix/Cytoperm Kit (BD) after antibody staining, followed by intracellular staining with a FITC-conjugated anti-pimonidazole antibody (1:100 dilution) and analyzed using flow cytometry.

Stimulation of cocultures of MDDC with CMV proteins

MDDCs from buffy coats provided by the “Centro de Transfusiones de Comunidad de Madrid” were generated from circulating monocytes as described (77). MDDCs were stimulated overnight with 4 μg/ml of a pp65 CMV peptide (PM-PP65-1, JPT) or DMSO as a control, in combination with 1 μg/ml of 2′3′-c-di-AM(PS)2 (Invivogen) STING agonist and 2.5 μg/ml of poly(I:C) (Sigma-Aldrich) TLR3 ligand as adjuvant. Activated MDDCs were cocultured with autologous total T lymphocytes isolated from peripheral blood mononuclear cells by negative selection using the Dynabeads Untouched Human T Cells Kit (Invitrogen) at a 1:3 ratio in RPMI medium containing antibiotics and 10% FBS supplemented with recombinant human interleukin-2 (50 IU/ml; Stem Cell Technologies) in the presence of 4 μg/ml of a pp65 CMV peptide and dasatinib or DMSO. T cells stimulated in parallel with PMA (50 ng/ml) and ionomycin (0.25 μg/ml) were included as a positive control of cytokine secretion. Autologous cocultures were incubated in normoxia or hypoxia in the presence of the peptides for 2 hours, and subsequently, brefeldin A (5 μg/ml), monensin (5 μg/ml), and anti-CD107a-APC (1:500 dilution) were added per well. After 4 hours, cells were stained and analyzed by flow cytometry. Normoxic cultures were maintained in at 37°C with 5% CO₂, following standard conditions, while hypoxic cultures were conducted in an ICO CO₂ incubator (Memmert GmbH, Germany) with 1% O₂ and 5% CO₂ at 37°C.

HEL-OVA immunization and ELISA

HEL-OVA immunization were performed as described (78) with some minor modifications. Mice were immunized with HEL-OVA (10 μg/ml) mixed with Montanide adjuvant (ISA 25 VG) at a 1:1 ratio. In brief, 24 hours after immunization, mice were treated with DMSO or dasatinib (50 mg/kg) by intraperitoneal injection for six consecutive days. On day 7 postinfection, mice were euthanized and blood samples were collected in serum-separating tubes. Samples were centrifuged at 10,000 rpm for 5 min to isolate serum, which was stored at −20°C until analysis. Standard enzyme-linked immunosorbent assay (ELISA) plates were coated with HEL-OVA (10 μg/ml) and incubated overnight at 4°C. Serum samples were diluted (1:50 to 1:150) in assay buffer and added to the plates in duplicates. Plates were then incubated with a biotinylated goat anti-mouse immunoglobulin M detection antibody (AbD serotec, catalog no. 102008), followed by horseradish peroxidase–conjugated streptavidin (BioLegend, catalog no. 405210). The optical density was measured at 450 nm using SpectraMax iD5.

Statistical analysis

Statistical significance was tested with paired and unpaired Student’s t test, one-way analysis of variance (ANOVA), or Mann-Whitney test using GraphPad Prism software v8. For each experiment, the test is specified in the corresponding figure legend. P values of <0.05 were considered statistically significant.

Acknowledgments

We thank M.J. Calzada for providing access to the hypoxia chamber, which was funded by the Redes de Biomedicina de la Comunidad de Madrid (S2022/BMD-7224).

Funding:

This work was supported by the San Salvatore Foundation (J.V.S and C.M.-C.); Swiss National Foundation grants 31003A_172994, 310030_200406, and 320030-236044 (J.V.S.); Spanish Instituto de Investigación Carlos III (ISCIII) grant PI21/01803 (C.M.-C.); ISCIII grant JR23/00062 cofunded by European Social Fund, “Investing in your future” (A.M.-J.); The Spanish Agencia Estatal de Investigación Generación de Conocimiento and Consolidation programs PID2021-127899OB-I00 and CNS2023-144841 (E.M.-G.); Gilead Biosciences grant GLD24/00117 (E.M.-G.); Infectious Diseases CIBER from ISCIII (CIBERINFECC) (C.M.-C. and E.M.-G.).; Spanish Ministry of Science Innovation and Universities, Beca de Formación de Profesorado Universitario (FPU) (I.T.); and Agencia Estatal de Investigación (MCIN/AEI/10.13039/501100011033) and ERDF “A way of making Europe” grant PID2020-116498RB-I00 (A.M.B.).

Author contributions:

D.C.C.: Conceptualization, investigation, writing—review and editing, methodology, validation, formal analysis, software, and visualization. A.M.-J.: Writing—original draft, conceptualization, investigation, methodology, validation, formal analysis, and visualization. I.T.: Writing—original draft, conceptualization, investigation, writing—review and editing, methodology, resources, validation, and formal analysis. C.V.P.: Investigation, methodology and formal analysis. S.S.L.: Investigation, methodology and formal analysis. I.E.: Investigation, methodology, and formal analysis. R.H.: Conceptualization, investigation, and formal analysis. A.M.B.: Investigation, writing—review and editing, and visualization. E.M.-G.: Conceptualization, investigation, and formal analysis. C.M.-C.: Writing—original draft, conceptualization, methodology, funding acquisition, supervision, and project administration. J.V.S.: Writing—original draft, conceptualization, writing—review and editing, methodology, funding acquisition, supervision, project administration, and visualization.

Competing interests:

The authors declare that they have no competing interests.

Data, code, and materials availability:

All data and code needed to evaluate and reproduce the results in the paper are present in the paper and/or the Supplementary Materials. This study did not generate new materials.

Supplementary Materials

The PDF file includes:

Figs. S1 to S4

Legend for movie S1

sciadv.aec5951_sm.pdf^{(928.1KB, pdf)}

Other Supplementary Material for this manuscript includes the following:

Movie S1

sciadv.aec5951_movie_s1.zip^{(7.8MB, zip)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figs. S1 to S4

Legend for movie S1

sciadv.aec5951_sm.pdf^{(928.1KB, pdf)}

Movie S1

sciadv.aec5951_movie_s1.zip^{(7.8MB, zip)}

Data Availability Statement

All data and code needed to evaluate and reproduce the results in the paper are present in the paper and/or the Supplementary Materials. This study did not generate new materials.

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PERMALINK

Dasatinib reversibly disrupts T cell motility and activation in lymph nodes

Daniela Claudino Carvoeiro

Ana Marcos-Jiménez

Ilya Tsukalov

Clara Viñas i Palou

Sergio Sanchez Luquin

Ihssane Eladnani

Raquel Hernanz

Ana M Briones

Enrique Martín-Gayo

Cecilia Muñoz-Calleja

Jens V Stein

Roles

Abstract

INTRODUCTION

RESULTS

Dasatinib impairs B but not T cell in vitro chemotaxis

Fig. 1. Dasatinib impairs B but not T cell in vitro migration to homeostatic chemokines.

Dasatinib induces a profound yet transient decrease in interstitial T and B cell motility

Fig. 2. Dasatinib causes a transient reduction in B and T cell in vivo motility.

Dasatinib induces an increase in hypoxia in peripheral LNs but not spleen

Fig. 3. Dasatinib triggers vasoconstriction and hypoxia in LNs.

In vitro dasatinib administration blocks DC-mediated CD8+ T cell activation at peak concentrations

Fig. 4. Impact of dasatinib on in vitro mouse CD8+ T cell activation.

Transient hypoxia does not inhibit early CD8+ T cell activation

Fig. 5. Transient hypoxia does not affect human CMV-specific CD8+ T cell activation.

Dasatinib administration delays but does not block DC-mediated CD8+ T cell activation

Fig. 6. Dasatinib delays DC vaccination-induced T cell activation in reactive LNs.

DISCUSSION

MATERIALS AND METHODS

Mice

Cells isolation and culture, and general reagents for in vitro and in vivo use

In vitro chemotaxis

Flow cytometry

Under agarose assays

Intravital two-photon microscopy

Infection model

Assessment of in vivo hypoxia using pimonidazole staining

Stimulation of cocultures of MDDC with CMV proteins

HEL-OVA immunization and ELISA

Statistical analysis

Acknowledgments

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Author contributions:

Competing interests:

Data, code, and materials availability:

Supplementary Materials

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Other Supplementary Material for this manuscript includes the following:

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

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RESOURCES

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In vitro dasatinib administration blocks DC-mediated CD8⁺ T cell activation at peak concentrations

Fig. 4. Impact of dasatinib on in vitro mouse CD8⁺ T cell activation.

Transient hypoxia does not inhibit early CD8⁺ T cell activation

Fig. 5. Transient hypoxia does not affect human CMV-specific CD8⁺ T cell activation.

Dasatinib administration delays but does not block DC-mediated CD8⁺ T cell activation