Introduction: Success rates of autologous T cell-based therapies such as CAR-T cell therapy, highly depend on T cell fitness. We have previously shown that T cells of CLL patients are functionally and metabolically impaired, limiting successful application of CAR-T cell therapy (van Bruggen et al., Blood, 2019). Although T cell exhaustion has been suggested as underlying mechanism, our observation that CLL T cell function can be restored by purifying CLL T cells argues against an exhausted state, and indicates that an as yet unknown CLL-derived factor is responsible for the acquired but reversible T-cell dysfunction (van Bruggen et al. Blood advances 2022). We aimed to analyze the impact of CLL on T cell function overtime in order to unravel the mechanism by which CLL cells disrupt T cell fitness.

Methods: Peripheral blood mononuclear cells (PBMCs) of healthy donors (HD) and untreated CLL patients and were analyzed by transcriptomics (CLL) and flow cytometry (HD and CLL). T cells were analyzed either directly after thawing or after a 2-day culture or over a time span of 16 days with or without αCD3/αCD28 antibodies.


Analysis of autologous T cell activation over-time revealed that T cell activation (CD25, and CD71) occurs in presence of CLL cells but in a significant delayed fashion while PD-1 expression remains elevated upon activation in CLL derived T cells compared to HD T cells (Fig. 1A). As a consequence, activation of CD19-CAR-T cells derived from CLL patients results in decreased proliferation, cytokine production and cytotoxicity in presence of autologous CLL cells. These findings, combined with our previous observations on improved T cell activation in absence of CLL cells, show that T cells in CLL are not (terminally) exhausted, and that a CLL-derived factor interferes with proper T-cell activation, leading to a delay in activation and impaired proliferation and cytotoxicity. We therefore set-out to identify the potential underlying CLL-derived factor(s).

In contrast to resting CLL cells, coculture with CD40-activated CLL cells improves CAR-T cell activation and proliferation (McKensie, Canc Res Comm 2022). We employed this model to decipher mechanisms of CLL-mediated T cell dysfunction. CD40-activation of CLL cells resulted in improved T-cell activation and proliferation upon αCD3/CD28 stimulation but only in a contact-dependent manner. Pre-treatment of CLL cells with the SRC-inhibitor dasatinib (100nM) abrogated the enhanced T-cell activation induced by CD40-activated CLL cells. Additional control experiments excluded direct effects of dasatinib on T cell function. Dasatinib did not reduce the CD40-mediated enhanced expression of co-stimulatory markers on CLL cells, indicating that lack of co-stimulation was not the sole explanation for CLL-mediated T cell dysfunction. Unbiased RNA-seq analyses of resting versus CD40-stimulated CLL cells treated with or without dasatinib and filtered for membrane-bound factors revealed the Sialic acid-binding Ig-like lectin 10 (Siglec-10) ligands CD24 and CD52 as potential candidates responsible for inhibiting T-cell function in CLL. Both at transcription and protein level, CD40-ligation resulted in suppressed expression of these ligands, which did not occur in presence of dasatinib. We also found that Siglec-10 expression is increased on CLL-T cells. These data suggest a role for Siglec-10 ligation in inhibition of the TCR signaling cascade. Indeed, blocking antibodies against CD24 and CD52 in the presence of autologous CLL cells improved T-cell activation, restored delayed activation (Fig. 1B), and repressed PD-1 expression.


We conclude that T cell dysfunction of CLL-derived T cells is reversible by blocking CD24- and CD52-ligation with Siglec-10. These proteins might represent targets for therapeutic intervention aimed at enhancing T-cell based therapies in CLL such as CAR-T cell therapy.

Kater:Abbvie, Astra Zeneca, BMS, Janssen, Roche/Genentech: Research Funding; Abbvie, Astra Zeneca, Janssen: Other: Speakers fee; Astra Zeneca, BMS, Roche/Gennetech, Janssen, Abbvie, LAVA: Membership on an entity's Board of Directors or advisory committees; Janssen, LAVA: Patents & Royalties: Pending; Amsterdam UMC, University of Amsterdam: Current Employment.

Author notes


Asterisk with author names denotes non-ASH members.

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