Leukemic cell-secreted interleukin-9 suppresses cytotoxic T cell-mediated killing in chronic lymphocytic leukemia.

The tumor microenvironment (TME) plays a central role in the pathogenesis of chronic lymphocytic leukemia (CLL), contributing to disease progression and chemoresistance. Leukemic cells shape the TME into a pro-survival and immunosuppressive niche through contact-dependent and contact-independent interactions with the cellular components of the TME. Immune synapse (IS) formation is defective in CLL. Here we asked whether soluble factors released by CLL cells contribute to their protection from cytotoxic T cell (CTL)-mediated killing by interfering with this process. We found that healthy CTLs cultured in media conditioned by leukemic cells from CLL patients or Eµ-TCL1 mice upregulate the exhaustion marker PD-1 and become unable to form functional ISs and kill target cells. These defects were more pronounced when media were conditioned by leukemic cells lacking p66Shc, a proapoptotic adapter whose deficiency has been implicated in disease aggressiveness both in CLL and in the Eµ-TCL1 mouse model. Multiplex ELISA assays showed that leukemic cells from Eµ-TCL1 mice secrete abnormally elevated amounts of CCL22, CCL24, IL-9 and IL-10, which are further upregulated in the absence of p66Shc. Among these, IL-9 and IL-10 were also overexpressed in leukemic cells from CLL patients, where they inversely correlated with residual p66Shc. Using neutralizing antibodies or the recombinant cytokines we show that IL-9, but not IL-10, mediates both the enhancement in PD-1 expression and the suppression of effector functions in healthy CTLs. Our results demonstrate that IL-9 secreted by leukemic cells negatively modulates the anti-tumor immune abilities of CTLs, highlighting a new suppressive mechanism and a novel potential therapeutical target in CLL.

Polo-like kinase 1 regulates immune synapse assembly and cytotoxic T cell function by driving microtubule dynamics.

Elimination of virally infected or tumoral cells is mediated by cytotoxic T cells (CTL). Upon antigen recognition, CTLs assemble a specialized signaling and secretory domain at the interface with their target, the immune synapse (IS). During IS formation, CTLs acquire a transient polarity, marked by re-orientation of the centrosome and microtubule cytoskeleton toward the IS, thus directing the transport and delivery of the lytic granules to the target cell. Based on the implication that the kinase Aurora A has a role in CTL function, we hypothesized that its substrate, the mitotic regulator Polo-like kinase 1 (PLK1), might participate in CTL IS assembly. We demonstrate that PLK1 is phosphorylated upon TCR triggering and polarizes to the IS. PLK1 silencing or inhibition results in impaired IS assembly and function, as witnessed by defective synaptic accumulation of T cell receptors (TCRs), as well as compromised centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing. This function is achieved by coupling early signaling to microtubule dynamics, a function pivotal for CTL-mediated cytotoxicity. These results identify PLK1 as a new player in CTL IS assembly and function.

SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly.

CTL-mediated killing of virally infected or malignant cells is orchestrated at the immune synapse (IS). We hypothesized that SARS-CoV-2 may target lytic IS assembly to escape elimination. We show that human CD8+ T cells upregulate the expression of ACE2, the Spike receptor, during differentiation to CTLs. CTL preincubation with the Wuhan or Omicron Spike variants inhibits IS assembly and function, as shown by defective synaptic accumulation of TCRs and tyrosine phosphoproteins as well as defective centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing and cytokine production. These defects were reversed by anti-Spike antibodies interfering with ACE2 binding and reproduced by ACE2 engagement by angiotensin II or anti-ACE2 antibodies, but not by the ACE2 product Ang (1-7). IS defects were also observed ex vivo in CTLs from COVID-19 patients. These results highlight a new strategy of immune evasion by SARS-CoV-2 based on the Spike-dependent, ACE2-mediated targeting of the lytic IS to prevent elimination of infected cells.

IFT20: An Eclectic Regulator of Cellular Processes beyond Intraflagellar Transport.

Initially discovered as the smallest component of the intraflagellar transport (IFT) system, the IFT20 protein has been found to be implicated in several unconventional mechanisms beyond its essential role in the assembly and maintenance of the primary cilium. IFT20 is now considered a key player not only in ciliogenesis but also in vesicular trafficking of membrane receptors and signaling proteins. Moreover, its ability to associate with a wide array of interacting partners in a cell-type specific manner has expanded the function of IFT20 to the regulation of intracellular degradative and secretory pathways. In this review, we will present an overview of the multifaceted role of IFT20 in both ciliated and non-ciliated cells.

A CVID-associated variant in the ciliogenesis protein CCDC28B disrupts immune synapse assembly.

Ciliogenesis proteins orchestrate vesicular trafficking pathways that regulate immune synapse (IS) assembly in the non-ciliated T-cells. We hypothesized that ciliogenesis-related genes might be disease candidates for common variable immunodeficiency with impaired T-cell function (T-CVID). We identified a heterozygous, predicted pathogenic variant in the ciliogenesis protein CCDC28B present with increased frequency in a large CVID cohort. We show that CCDC28B participates in IS assembly by regulating polarized T-cell antigen receptor (TCR) recycling. This involves the CCDC28B-dependent, FAM21-mediated recruitment of the actin regulator WASH to retromer at early endosomes to promote actin polymerization. The CVID-associated CCDC28BR25W variant failed to interact with FAM21, leading to impaired synaptic TCR recycling. CVID T cells carrying the ccdc28b 211 C > T allele displayed IS defects mapping to this pathway that were corrected by overexpression of the wild-type allele. These results identify a new disease gene in T-CVID and pinpoint CCDC28B as a new player in IS assembly.

The Bardet-Biedl syndrome complex component BBS1 controls T cell polarity during immune synapse assembly.

Components of the intraflagellar transport (IFT) system that regulates the assembly of the primary cilium are co-opted by the non-ciliated T cell to orchestrate polarized endosome recycling and to sustain signaling during immune synapse formation. Here, we investigated the potential role of Bardet-Biedl syndrome 1 protein (BBS1), an essential core component of the BBS complex that cooperates with the IFT system in ciliary protein trafficking, in the assembly of the T cell synapse. We demonstrated that BBS1 allows for centrosome polarization towards the immune synapse. This function is achieved through the clearance of centrosomal F-actin and its positive regulator WASH1 (also known as WASHC1), a process that we demonstrated to be dependent on the proteasome. We show that BBS1 regulates this process by coupling the 19S proteasome regulatory subunit to the microtubule motor dynein for its transport to the centrosome. Our data identify the ciliopathy-related protein BBS1 as a new player in T cell synapse assembly that functions upstream of the IFT system to set the stage for polarized vesicular trafficking and sustained signaling. This article has an associated First Person interview with the first author of the paper.

The Intraflagellar Transport Protein IFT20 Recruits ATG16L1 to Early Endosomes to Promote Autophagosome Formation in T Cells.

Lymphocyte homeostasis, activation and differentiation crucially rely on basal autophagy. The fine-tuning of this process depends on autophagy-related (ATG) proteins and their interaction with the trafficking machinery that orchestrates the membrane rearrangements leading to autophagosome biogenesis. The underlying mechanisms are as yet not fully understood. The intraflagellar transport (IFT) system, known for its role in cargo transport along the axonemal microtubules of the primary cilium, has emerged as a regulator of autophagy in ciliated cells. Growing evidence indicates that ciliogenesis proteins participate in cilia-independent processes, including autophagy, in the non-ciliated T cell. Here we investigate the mechanism by which IFT20, an integral component of the IFT system, regulates basal T cell autophagy. We show that IFT20 interacts with the core autophagy protein ATG16L1 and that its CC domain is essential for its pro-autophagic activity. We demonstrate that IFT20 is required for the association of ATG16L1 with the Golgi complex and early endosomes, both of which have been identified as membrane sources for phagophore elongation. This involves the ability of IFT20 to interact with proteins that are resident at these subcellular localizations, namely the golgin GMAP210 at the Golgi apparatus and Rab5 at early endosomes. GMAP210 depletion, while leading to a dispersion of ATG16L1 from the Golgi, did not affect basal autophagy. Conversely, IFT20 was found to recruit ATG16L1 to early endosomes tagged for autophagosome formation by the BECLIN 1/VPS34/Rab5 complex, which resulted in the local accumulation of LC3. Hence IFT20 participates in autophagosome biogenesis under basal conditions by regulating the localization of ATG16L1 at early endosomes to promote autophagosome biogenesis. These data identify IFT20 as a new regulator of an early step of basal autophagy in T cells.

Enhanced IL-9 secretion by p66Shc-deficient CLL cells modulates the chemokine landscape of the stromal microenvironment.

The stromal microenvironment is central to chronic lymphocytic leukemia (CLL) pathogenesis. How leukemic cells condition the stroma to enhance its chemoattractant properties remains elusive. Here, we show that mouse and human CLL cells promote the contact-independent stromal expression of homing chemokines. This function was strongly enhanced in leukemic cells from Eµ-TCL1 mice lacking the pro-oxidant p66Shc adaptor, which develop an aggressive disease with organ infiltration. We identified interleukin-9 (IL-9) as the soluble factor, negatively modulated by p66Shc, that is responsible for the chemokine-elevating activity of leukemic cells on stromal cells. IL-9 blockade in Eµ-TCL1/p66Shc-/- mice resulted in a decrease in the nodal expression of homing chemokines, which correlated with decreased leukemic cell invasiveness. IL-9 levels were found to correlate inversely with residual p66Shc in p66Shc-deficient human CLL cells (n = 52 patients). p66Shc reconstitution in CLL cells normalized IL-9 expression and neutralized their chemokine-elevating activity. Notably, high IL-9 expression in CLL cells directly correlates with lymphadenopathy, liver infiltration, disease severity, and overall survival, emerging as an independent predictor of disease outcome. Our results demonstrate that IL-9 modulates the chemokine landscape in the stroma and that p66Shc, by regulating IL-9 expression, fine tunes the ability of leukemic cells to shape the microenvironment, thereby contributing to CLL pathogenesis.

Regulation of Selective B Cell Autophagy by the Pro-oxidant Adaptor p66SHC.

p66SHC is a pro-oxidant member of the SHC family of protein adaptors that acts as a negative regulator of cell survival. In lymphocytes p66SHC exploits both its adaptor and its reactive oxygen species (ROS)-elevating function to antagonize mitogenic and survival signaling and promote apoptosis. As a result, p66SHC deficiency leads to the abnormal expansion of peripheral T and B cells and lupus-like autoimmunity. Additionally, a defect in p66SHC expression is a hallmark of B cell chronic lymphocytic leukemia, where it contributes to the accumulation of long-lived neoplastic cells. We have recently provided evidence that p66SHC exerts a further layer of control on B cell homeostasis by acting as a new mitochondrial LC3-II receptor to promote the autophagic demise of dysfunctional mitochondria. Here we discuss this finding in the context of the autophagic control of B cell homeostasis, development, and differentiation in health and disease.

Optimization of Organotypic Cultures of Mouse Spleen for Staining and Functional Assays.

By preserving cell viability and three-dimensional localization, organotypic culture stands out among the newest frontiers of cell culture. It has been successfully employed for the study of diseases among which neoplasias, where tumoral cells take advantage of the surrounding stroma to promote their own proliferation and survival. Organotypic culture acquires major importance in the context of the immune system, whose cells cross-talk in a complex and dynamic fashion to elicit productive responses. However, organotypic culture has been as yet poorly developed for and applied to primary and secondary lymphoid organs. Here we describe in detail the development of a protocol suitable for the efficient cutting of mouse spleen, which overcomes technical difficulties related to the peculiar organ texture, and for optimized organotypic culture of spleen slices. Moreover, we used microscopy, immunofluorescence, flow cytometry, and qRT-PCR to demonstrate that the majority of cells residing in spleen slices remain alive and maintain their original location in the organ architecture for several days after cutting. The development of this protocol represents a significant technical improvement in the study of the lymphoid microenvironment in both physiological and pathological conditions involving the immune system.

The intraflagellar transport protein IFT20 controls lysosome biogenesis by regulating the post-Golgi transport of acid hydrolases.

The assembly and function of the primary cilium depends on multimolecular intraflagellar transport (IFT) complexes that shuttle their cargo along the axonemal microtubules through their interaction with molecular motors. The IFT system has been moreover recently implicated in a reciprocal interplay between autophagy and ciliogenesis. We have previously reported that IFT20 and other components of the IFT complexes participate in the assembly of the immune synapse in the non-ciliated T cell, suggesting that other cellular processes regulated by the IFT system in ciliated cells, including autophagy, may be shared by cells lacking a cilium. Starting from the observation of a defect in autophagic clearance and an accumulation of lipid droplets in IFT20-deficient T cells, we show that IFT20 is required for lysosome biogenesis and function by controlling the lysosomal targeting of acid hydrolases. This function involves its ability to regulate the retrograde traffic of the cation-independent mannose-6-phosphate receptor (CI-MPR) to the trans-Golgi network, which is achieved by coupling recycling CI-MPRs to the microtubule motor dynein. Consistent with the lysosomal defect, an upregulation of the TFEB-dependent expression of the lysosomal gene network can be observed in IFT20-deficient cells, which is associated with defective tonic T-cell antigen receptor signaling and mTOR activity. We additionally show that the lysosome-related function of IFT20 extends to non-ciliated cells other than T cells, as well as to ciliated cells. Our findings provide the first evidence that a component of the IFT system that controls ciliogenesis is implicated in the biogenesis of lysosomes.

Emerging Roles of the Intraflagellar Transport System in the Orchestration of Cellular Degradation Pathways.

Ciliated cells exploit a specific transport system, the intraflagellar transport (IFT) system, to ensure the traffic of molecules from the cell body to the cilium. However, it is now clear that IFT activity is not restricted to cilia-related functions. This is strikingly exemplified by the observation that IFT proteins play important roles in cells lacking a primary cilium, such as lymphocytes. Indeed, in T cells the IFT system regulates the polarized transport of endosome-associated T cell antigen receptors and signaling mediators during assembly of the immune synapse, a specialized interface that forms on encounter with a cognate antigen presenting cell and on which T cell activation and effector function crucially depend. Cellular degradation pathways have recently emerged as new extraciliary functions of the IFT system. IFT proteins have been demonstrated to regulate autophagy in ciliated cells through their ability to recruit the autophagy machinery to the base of the cilium. We have now implicated the IFT component IFT20 in another central degradation process that also controls the latest steps in autophagy, namely lysosome function, by regulating the cation-independent mannose-6-phosphate receptor (CI-MPR)-dependent lysosomal targeting of acid hydrolases. This involves the ability of IFT20 to act as an adaptor coupling the CI-MPR to dynein for retrograde transport to the trans-Golgi network. In this short review we will discuss the emerging roles of IFT proteins in cellular degradation pathways.

A T Cell Suppressive Circuitry Mediated by CD39 and Regulated by ShcC/Rai Is Induced in Astrocytes by Encephalitogenic T Cells.

Multiple sclerosis is an autoimmune disease caused by autoreactive immune cell infiltration into the central nervous system leading to inflammation, demyelination, and neuronal loss. While myelin-reactive Th1 and Th17 are centrally implicated in multiple sclerosis pathogenesis, the local CNS microenvironment, which is shaped by both infiltrated immune cells and central nervous system resident cells, has emerged a key player in disease onset and progression. We have recently demonstrated that ShcC/Rai is as a novel astrocytic adaptor whose loss in mice protects from experimental autoimmune encephalomyelitis. Here, we have explored the mechanisms that underlie the ability of Rai-/- astrocytes to antagonize T cell-dependent neuroinflammation. We show that Rai deficiency enhances the ability of astrocytes to upregulate the expression and activity of the ectonucleotidase CD39, which catalyzes the conversion of extracellular ATP to the immunosuppressive metabolite adenosine, through both contact-dependent and-independent mechanisms. As a result, Rai-deficient astrocytes acquire an enhanced ability to suppress T-cell proliferation, which involves suppression of T cell receptor signaling and upregulation of the inhibitory receptor CTLA-4. Additionally, Rai-deficient astrocytes preferentially polarize to the neuroprotective A2 phenotype. These results identify a new mechanism, to which Rai contributes to a major extent, by which astrocytes modulate the pathogenic potential of autoreactive T cells.

p66Shc deficiency in the Eµ-TCL1 mouse model of chronic lymphocytic leukemia enhances leukemogenesis by altering the chemokine receptor landscape.

The Shc family adaptor p66Shc acts as a negative regulator of proliferative and survival signals triggered by the B-cell receptor and, by enhancing the production of reactive oxygen species, promotes oxidative stress-dependent apoptosis. Additionally, p66Shc controls the expression and function of chemokine receptors that regulate lymphocyte traffic. Chronic lymphocytic leukemia cells have a p66Shc expression defect which contributes to their extended survival and correlates with poor prognosis. We analyzed the impact of p66Shc ablation on disease severity and progression in the Eµ-TCL1 mouse model of chronic lymphocytic leukemia. We showed that Eµ-TCL1/p66Shc-/- mice developed an aggressive disease that had an earlier onset, occurred at a higher incidence and led to earlier death compared to that in Eµ-TCL1 mice. Eµ-TCL1/p66Shc-/- mice displayed substantial leukemic cell accumulation in both nodal and extranodal sites. The target organ selectivity correlated with upregulation of chemokine receptors whose ligands are expressed therein. This also applied to chronic lymphocytic leukemia cells, where chemokine receptor expression and extent of organ infiltration were found to correlate inversely with these cells' level of p66Shc expression. p66Shc expression declined with disease progression in Eµ-TCL1 mice and could be restored by treatment with the Bruton tyrosine kinase inhibitor ibrutinib. Our results highlight p66Shc deficiency as an important factor in the progression and severity of chronic lymphocytic leukemia and underscore p66Shc expression as a relevant therapeutic target.

The immunological synapse as a pharmacological target.

The development of T cell mediated immunity relies on the assembly of a highly specialized interface between T cell and antigen presenting cell (APC), known as the immunological synapse (IS). IS assembly is triggered when the T cell receptor (TCR) binds to specific peptide antigen presented in association to the major histocompatibility complex (MHC) by the APC, and is followed by the spatiotemporal dynamic redistribution of TCR, integrins, co-stimulatory receptors and signaling molecules, allowing for the fine-tuning and integration of the signals that lead to T cell activation. The knowledge acquired to date about the mechanisms of IS assembly underscores this structure as a robust pharmacological target. The activity of molecules involved in IS assembly and function can be targeted by specific compounds to modulate the immune response in a number of disorders, including cancers and autoimmune diseases, or in transplanted patients. Here, we will review the state-of-the art of the current therapies which exploit the IS to modulate the immune response.

Compartmentalized Cyclic AMP Production by the Bordetella pertussis and Bacillus anthracis Adenylate Cyclase Toxins Differentially Affects the Immune Synapse in T Lymphocytes.

A central feature of the immune synapse (IS) is the tight compartmentalization of membrane receptors and signaling mediators that is functional for its ability to coordinate T cell activation. Second messengers centrally implicated in this process, such as Ca2+ and diacyl glycerol, also undergo compartmentalization at the IS. Current evidence suggests a more complex scenario for cyclic AMP (cAMP), which acts both as positive and as negative regulator of T-cell antigen receptor (TCR) signaling and which, as such, must be subjected to a tight spatiotemporal control to allow for signaling at the IS. Here, we have used two bacterial adenylate cyclase toxins that produce cAMP at different subcellular localizations as the result of their distinct routes of cell invasion, namely Bordetella pertussis CyaA and Bacillus anthracis edema toxin (ET), to address the ability of the T cell to confine cAMP to the site of production and to address the impact of compartmentalized cAMP production on IS assembly and function. We show that CyaA, which produces cAMP close to the synaptic membrane, affects IS stability by modulating not only the distribution of LFA-1 and its partners talin and L-plastin, as previously partly reported but also by promoting the sustained synaptic accumulation of the A-kinase adaptor protein ezrin and protein kinase A while suppressing the ß-arrestin-mediated recruitment of phosphodiesterase 4B. These effects are dependent on the catalytic activity of the toxin and can be reproduced by treatment with a non-hydrolyzable cAMP analog. Remarkably, none of these effects are elicited by ET, which produces cAMP at a perinuclear localization, despite its ability to suppress TCR signaling and T cell activation through its cAMP-elevating activity. These results show that the IS responds solely to local elevations of cAMP and provide evidence that potent compartmentalization mechanisms are operational in T cells to contain cAMP close to the site of production, even when produced at supraphysiological levels.

Transcellular communication at the immunological synapse: a vesicular traffic-mediated mutual exchange.

The cell's ability to communicate with the extracellular environment, with other cells, and with itself is a crucial feature of eukaryotic organisms. In the immune system, T lymphocytes assemble a specialized structure upon contact with antigen-presenting cells bearing a peptide-major histocompatibility complex ligand, known as the immunological synapse (IS). The IS has been extensively characterized as a signaling platform essential for T-cell activation. Moreover, emerging evidence identifies the IS as a device for vesicular traffic-mediated cell-to-cell communication as well as an active release site of soluble molecules. Here, we will review recent advances in the role of vesicular trafficking in IS assembly and focused secretion of microvesicles at the synaptic area in naïve T cells and discuss the role of the IS in transcellular communication.

Vesicular Trafficking to the Immune Synapse: How to Assemble Receptor-Tailored Pathways from a Basic Building Set.

The signals that orchestrate T-cell activation are coordinated within a highly organized interface with the antigen-presenting cell (APC), known as the immune synapse (IS). IS assembly depends on T-cell antigen receptor engagement by a specific peptide antigen-major histocompatibility complex ligand. This primary event leads to polarized trafficking of receptors and signaling mediators associated with recycling endosomes to the cellular interface, which contributes to IS assembly as well as signal termination and favors information transfer from T cells to APCs. Here, we will review recent advances on the vesicular pathways implicated in IS assembly and maintenance, focusing on the spatiotemporal regulation of the traffic of specific receptors by Rab GTPases. Based on accumulating evidence that the IS is a functional homolog of the primary cilium, which coordinates several central signaling pathways in ciliated cells, we will also discuss the similarities in the mechanisms regulating vesicular trafficking to these specialized membrane domains.

The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse.

IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, regulates immune synapse assembly in the non-ciliated T-cell by promoting T-cell receptor (TCR) recycling. Here, we have addressed the role of Rab8 (for which there are two isoforms Rab8a and Rab8b), a small GTPase implicated in ciliogenesis, in TCR traffic to the immune synapse. We show that Rab8, which colocalizes with IFT20 in Rab11(+) endosomes, is required for TCR recycling. Interestingly, as opposed to in IFT20-deficient T-cells, TCR(+) endosomes polarized normally beneath the immune synapse membrane in the presence of dominant-negative Rab8, but were unable to undergo the final docking or fusion step. This could be accounted for by the inability of the vesicular (v)-SNARE VAMP-3 to cluster at the immune synapse in the absence of functional Rab8, which is responsible for its recruitment. Of note, and similar to in T-cells, VAMP-3 interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of the protein smoothened. The results identify Rab8 as a new player in vesicular traffic to the immune synapse and provide insight into the pathways co-opted by different cell types for immune synapse assembly and ciliogenesis.