T cells in CLL: lost in migration

In this issue of Blood, Ramsay et al¹  unravel a new mechanism of immune subversion induced by chronic lymphocytic leukemia (CLL) cells to perturb chemokine-oriented migration of both CD4⁺ and CD8⁺ T cells, and they propose an original cereblon-dependent effect of the immunomodulatory drug lenalidomide.

During the journey of homing to lymph nodes, T cells undergo a stepwise adhesion process that culminates in a strong, dependent adhesion of lymphocyte function–associated antigen-1 (LFA-1, also known as α2βL integrin and as CD11a) to intercellular adhesion molecule-1 (ICAM-1, or CD54) expressed at the luminal surface of high endothelial venules (HEVs). The activation of LFA-1 through diverse receptors, such as chemokine receptors, results in the transformation of the integrin from a bent, resting form (with low affinity for CD54) to an extended conformation (with high affinity), a process called “outside-in signaling.” This highly regulated LFA-1 activation is essential for T-cell migration and the formation of an immunological synapse.^2,3  Chemokine signaling is also critical to direct T-cell polarity and migration through the activation of specific intracellular pathways, among which small guanosine triphosphatases (GTPases) of the Rho family (RhoA, Rac1, Cdc42) and polarity proteins interplay in a highly orchestrated manner. During chemotaxis, GTPases regulate actin polymerization, which is coordinated with LFA-1 redistribution to the leading edge of the cell. The Ras-proximity protein Rap1 activates Rac1, which promotes actin reorganization, triggering lamellipodium formation and the crawling of the T cell toward the chemokine gradient (chemotaxis).

In the context of CLL, tumor cells fail to form stable cognate interactions with CD4⁺ and CD8⁺ T cells, due in part to an impaired clustering and activation of LFA-1 at the interface between the effector (T lymphocyte) and the target cells (CLL cell, or host antigen-presenting cell).⁴  CLL cells indeed induce profound modifications of T-cell transcriptomes,⁵  with specific emphasis on cytoskeletal regulators (functionally leading to decreased actin remodeling and nonpolarized degranulation). This further jeopardizes the formation of effective immune synapses, leading to a clinically relevant immunosuppressive state, but also generating a defect in host antitumor responses. CLL cells express surface molecules that act as ligands to co-opt inhibitory receptors that impair T-cell actin dynamics, in part through the regulation of small GTPases of the Rho family.⁶  As an immunomodulatory drug with clinical benefit in patients with CLL, lenalidomide induces the downregulation of CLL ligands and restores the stabilization of effective immune synapses (though a direct effect on LFA-1 is currently unclear).

This article by Ramsay et al is in line with their previous contributions to the field of immunosuppression resulting from CLL:T cell contacts, but they add further insight into a possibly more-general immune evasion mechanism in cancer. Leukemic cells induce a T-cell LFA-1–dependent adhesion/migration defect that is mediated by dysregulated Rho GTPase signaling. By using migration on CD54 toward a gradient of CXCL12 (SDF-1) or CCL19, ligands for chemokine receptors CXCR4 and CCR7, respectively, the authors first demonstrate a contact-dependent defect in polarized migration of CLL T cells as compared with age-matched controls (despite no evident discrepancies between chemokine receptor expression levels across samples). Then the differential activation status of LFA-1 is investigated by flow cytometry and confocal microscopy, with conformation-specific antibodies, to confirm CLL T cells preferentially express low affinity LFA-1 as compared with control T cells and, as a consequence, display decreased size and strength of contacts between LFA-1 and CD54 (as assessed with interference reflection microscopy). This lack of an active form of LFA-1 at the surface of T cells is suspected to reflect impaired outside-in signaling, because manganese (which bypasses chemokine signals) can restore strong LFA-1/CD54 interactions in CLL T cells. To focus more precisely on that, the authors use a pharmacological and small interfering RNA (siRNA) approach (to inhibit RhoA and Rac1) and the transfection of active mutants of Cdc42, the Rho GTPase that is transcriptionally increased in CLL T cells.⁵  Coculture with CLL cells induces activation of Cdc42 and impaired activation of RhoA and Rac1 in T cells (as measured with colorimetric assays, not pull-down assays). The observed reduced contacts between LFA-1 and CD54 in CLL T cells can be reproduced in healthy T cells by using the transfection of active Cdc42 and the inhibition of RhoA/Rac1, without the presence of CLL cells. As a readout for the dysregulated equilibrium between Rho GTPases and the expression of low-affinity conformation of LFA-1 in CLL T cells, confocal microscopy demonstrates a decreased rate of membrane-bound Rap1, a critical step to allow open conformation (high affinity) of LFA-1 after chemokine receptor activation, but also of phosphorylated myosin light chain (a downstream effector of Rho GTPases). The nature of the receptor/ligand(s) promoting the adhesion/migration defect in CLL T cells is unknown, and it may be different from those that hamper immune synapse polarization.⁶ 

The authors confirm that lenalidomide treatment re-equilibrates Rac1, RhoA, and Cdc42 levels of activity in CLL T cells and rescues LFA-1 function (without the direct effect being evaluable on chemokine receptor expression at the surface of T cells). Depicted for the first time, this effect on migration relies on cereblon expression in T cells, as siRNA directed against this classical lenalidomide intracellular target completely blocks the rescuing effect on adhesion and migration.

Cancer immune evasion mechanisms are important steps to promoting disease progression in CLL but also in other cancers. This original tumor-derived T-cell adhesion/migration defect compromises recruitment of T-cell subsets by HEV and infiltration in lymph nodes, and therefore the success of cell-based immunotherapeutic strategies. Besides this migration defect, the disorganized nodal architecture that results from diffuse CLL cell infiltration may also constitute a “physical barrier” preventing T-cell trafficking within lymph nodes.⁷  It is also widely admitted that, within the CLL niches (pseudofollicles), close interactions with diverse bystander stromal cells or CD4⁺ T cells provide prosurvival and immune escape signals to proliferating CLL cells. A strong challenge for any immunomodulatory therapy now is, can we improve recruitment of effective, nonexhausted⁸  T cells within lymph nodes, and make them find their way to and kill tumoral targets (through enhanced immune synapses), without getting hijacked by CLL cells to become bystander?