Challenges and next steps in the advancement of immunotherapy: summary of the 2018 and 2020 National Cancer Institute workshops on cell-based immunotherapy for solid tumors.

Cell-based immunotherapies have had remarkable success in the clinic, specifically in the treatment of hematologic malignancies. However, these strategies have had limited efficacy in patients with solid tumors. To better understand the challenges involved, the National Cancer Institute (NCI) convened an initial workshop with immuno-oncology thought leaders in December 2018 and a follow-up workshop in December 2020. The goals of the NCI workshops on cell-based immunotherapy for solid tumors were to discuss the current state of the field of cell-based immunotherapy, obtain insights into critical knowledge gaps, and identify ways in which NCI could facilitate progress. At both meetings, subjects emphasized four main types of challenges in further developing cell-based immunotherapy for patients with solid tumors: scientific, technical, clinical, and regulatory. The scientific barriers include selecting appropriate targets, ensuring adequate trafficking of cell therapy products to tumor sites, overcoming the immunosuppressive tumor microenvironment, and identifying appropriate models for these investigations. While mouse models may provide some useful data, the majority of those that are commonly used are immunodeficient and unable to fully recapitulate the immune response in patients. There is therefore a need for enhanced support of small early-phase human clinical studies, preferably with adaptive trial designs, to provide proof of concept for novel cell therapy approaches. Furthermore, the requirements for manufacturing, shipping, and distributing cell-based therapies present technical challenges and regulatory questions, which many research institutions are not equipped to address. Overall, workshop subjects identified key areas where NCI support might help the research community in driving forward innovation and clinical utility: 1) provide focused research support on topics such as tumor target selection, immune cell fitness and persistence, cell trafficking, and the immunosuppressive tumor microenvironment; 2) support the rapid translation of preclinical findings into proof of concept clinical testing, harmonize clinical trial regimens, and facilitate early trial data sharing (including negative results); 3) expand manufacturing support for cell therapies, including vectors and reagents, and provide training programs for technical staff; and 4) develop and share standard operating procedures for cell handling and analytical assays, and work with the Food and Drug Administration to harmonize product characterization specifications.

Expression of a TMC6-TMC8-CIB1 heterotrimeric complex in lymphocytes is regulated by each of the components.

The TMC genes encode a set of homologous transmembrane proteins whose functions are not well understood. Biallelic mutations in either TMC6 or TMC8 are detected in more than half of cases of the pre-malignant skin disease epidermodysplasia verruciformis (EV). It is controversial whether EV induced by mutations in TMC6 or TMC8 originates from keratinocyte or lymphocyte defects. Quantification of TMC6 and TMC8 RNA levels in various organs revealed that lymphoid tissues have the highest levels of expression of both genes, and custom antibodies confirmed protein expression in mouse lymphocytes. To study the function of these proteins we generated mice with targeted deletion mutant alleles of Tmc6 or Tmc8 Either TMC6 or TMC8 deficiency induced a reduction in apparent molecular weight and/or amount of the other TMC molecule. Co-immunoprecipitation experiments indicated that TMC6 and TMC8 formed a protein complex in mouse and human T cells. MS and biochemical analysis demonstrated that TMC6 and TMC8 additionally interacted with the CIB1 protein to form TMC6-TMC8-CIB1 trimers. We demonstrated that TMC6 and TMC8 regulated CIB1 levels by protecting CIB1 from ubiquitination and proteasomal degradation. Reciprocally, CIB1 was needed for stabilizing TMC6 and TMC8 levels. These results suggest why inactivating mutations in any of the three human genes leads to similar clinical presentations. We also demonstrated that TMC6 and TMC8 levels are drastically lower and the proteins are less active in regulating CIB1 in keratinocytes than in T cells. Our study suggests that defects in lymphocytes may contribute to the etiology and pathogenesis of EV.

Report on the 2018 Cancer, Autoimmunity, and Immunology Conference.

With the increased use of cancer immunotherapy, a number of immune-related adverse events (irAEs) are being identified. These irAEs can be compared with known autoimmune disorders in similar tissues, with important similarities and differences. Understanding the etiology of irAEs may bring to light concepts applicable to immune responses in cancer, autoimmunity, and infectious disease. This immunobiology is especially relevant to cancer patients with preexisting allogeneic transplants or autoimmune disease who are undergoing cancer immunotherapy. To address these facets of cancer immunotherapy, academic leaders from these various disciplines discussed current irAE basic and clinical research, irAE diagnosis and management, and the need for biomarkers and algorithms to identify individuals at risk for irAEs at a conference jointly sponsored by the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of Arthritis and Musculoskeletal and Skin Diseases in Bethesda, MD, on March 22-23, 2018. Mechanisms and models to characterize irAEs, standardize protocols, store biospecimens, and capture and analyze irAE data were also reviewed during the inaugural Cancer, Autoimmunity, and Immunology Conference. This summary highlights cancer immunotherapy-induced irAEs, the challenges ahead, and the opportunities for greater understanding of autoimmune conditions.

Unexpected Cartilage Phenotype in CD4-Cre-Conditional SOS-Deficient Mice.

RAS signaling is central to many cellular processes and SOS proteins promote RAS activation. To investigate the role of SOS proteins in T cell biology, we crossed Sos1f/fSos2-/- mice to CD4-Cre transgenic mice. We previously reported an effect of these mutations on T cell signaling and T cell migration. Unexpectedly, we observed nodules on the joints of greater than 90% of these mutant mice at 5 months of age, especially on the carpal joints. As the mice aged further, some also displayed joint stiffness, hind limb paralysis, and lameness. Histological analysis indicated that the abnormal growth in joints originated from dysplastic chondrocytes. Second harmonic generation imaging of the carpal nodules revealed that nodules were encased by rich collagen fibrous networks. Nodules formed in mice also deficient in RAG2, indicating that conventional T cells, which undergo rearrangement of the T cell antigen receptor, are not required for this phenotype. CD4-Cre expression in a subset of cells, either immune lineage cells (e.g., non-conventional T cells) or non-immune lineage cells (e.g., chondrocytes) likely mediates the dramatic phenotype observed in this study. Disruptions of genes in the RAS signaling pathway are especially likely to cause this phenotype. These results also serve as a cautionary tale to those intending to use CD4-Cre transgenic mice to specifically delete genes in conventional T cells.

Hierarchical nanostructure and synergy of multimolecular signalling complexes.

Signalling complexes are dynamic, multimolecular structures and sites for intracellular signal transduction. Although they play a crucial role in cellular activation, current research techniques fail to resolve their structure in intact cells. Here we present a multicolour, photoactivated localization microscopy approach for imaging multiple types of single molecules in fixed and live cells and statistical tools to determine the nanoscale organization, topology and synergy of molecular interactions in signalling complexes downstream of the T-cell antigen receptor. We observe that signalling complexes nucleated at the key adapter LAT show a hierarchical topology. The critical enzymes PLCγ1 and VAV1 localize to the centre of LAT-based complexes, and the adapter SLP-76 and actin molecules localize to the periphery. Conditional second-order statistics reveal a hierarchical network of synergic interactions between these molecules. Our results extend our understanding of the nanostructure of signalling complexes and are relevant to studying a wide range of multimolecular complexes.

miR-155 Controls Lymphoproliferation in LAT Mutant Mice by Restraining T-Cell Apoptosis via SHIP-1/mTOR and PAK1/FOXO3/BIM Pathways.

Linker for Activation of T cells (LAT) is an adapter protein that is essential for T cell function. Knock-in mice with a LAT mutation impairing calcium flux develop a fatal CD4+ lymphoproliferative disease. miR-155 is a microRNA that is correlated with hyperproliferation in a number of cancers including lymphomas and leukemias and is overexpressed in mutant LAT T cells. To test whether miR-155 was merely indicative of T cell activation or whether it contributes to lymphoproliferative disease in mutant LAT mice, we interbred LAT mutant and miR-155-deficient mice. miR-155 deficiency markedly inhibited lymphoproliferative disease by stimulating BIM-dependent CD4+ T cell apoptosis, even though ERK activation and T cell proliferation were increased in double mutant CD4+ T cells. Bim/Bcl2l11 expression is activated by the forkhead transcription factor FOXO3. Using miR-155-deficient, LAT mutant T cells as a discovery tool, we found two connected pathways that impact the nuclear translocation and activation of FOXO3 in T cells. One pathway is mediated by the inositide phosphatase SHIP-1 and the serine/threonine kinases AKT and PDK1. The other pathway involves PAK1 and JNK kinase activation. We define crosstalk between the two pathways via the kinase mTOR, which stabilizes PAK1. This study establishes a role for PAK1 in T cell apoptosis, which contrasts to its previously identified role in T cell proliferation. Furthermore, miR-155 regulates the delicate balance between PAK1-mediated proliferation and apoptosis in T cells impacting lymphoid organ size and function.

Absence of both Sos-1 and Sos-2 in peripheral CD4(+) T cells leads to PI3K pathway activation and defects in migration.

Sos-1 and Sos-2 are ubiquitously expressed Ras-guanine exchange factors involved in Erk-MAP kinase pathway activation. Using mice lacking genes encoding Sos-1 and Sos-2, we evaluated the role of these proteins in peripheral T-cell signaling and function. Our results confirmed that TCR-mediated Erk activation in peripheral CD4(+) T cells does not depend on Sos-1 and Sos-2, although IL-2-mediated Erk activation does. Unexpectedly, however, we show an increase in AKT phosphorylation in Sos-1/2dKO CD4(+) T cells upon TCR and IL-2 stimulation. Activation of AKT was likely a consequence of increased recruitment of PI3K to Grb2 upon TCR and/or IL-2 stimulation in Sos-1/2dKO CD4(+) T cells. The increased activity of the PI3K/AKT pathway led to downregulation of the surface receptor CD62L in Sos-1/2dKO T cells and a subsequent impairment in T-cell migration.

Diacylglycerol kinase ζ limits the generation of natural regulatory T cells.

Natural regulatory T (nT(reg)) cells are important for maintaining tolerance to self- and foreign antigens, and they are thought to develop from thymocytes that receive strong T cell receptor (TCR)-mediated signals in the thymus. TCR engagement leads to the activation of phospholipase C-γ1, which generates the lipid second messenger diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate. We used mice that lack the ζ isoform of DAG kinase (DGKζ), which metabolizes DAG to terminate its signaling, to enhance TCR-mediated signaling and identify critical signaling events in nT(reg) cell development. Loss of DGKζ resulted in increased numbers of thymic CD25(+)Foxp3(-)CD4(+) nT(reg) cell precursors and Foxp3(+)CD4(+) nT(reg) cells in a cell-autonomous manner. DGKζ-deficient T cells exhibited increased nuclear translocation of the nuclear factor κB subunit c-Rel, as well as enhanced extracellular signal-regulated kinase (ERK) phosphorylation in response to TCR stimulation, suggesting that these downstream pathways may contribute to nT(reg) cell development. Indeed, reducing c-Rel abundance or blocking ERK phosphorylation abrogated the increased generation of nTreg cells by DGKζ-deficient thymocytes. The extent of ERK phosphorylation correlated with TCR-mediated acquisition of Foxp3 in immature thymocytes in vitro. Furthermore, the development of nT(reg) cells was augmented in mice in which ERK activation was selectively enhanced in T cells. Together, these data suggest that DGKζ regulates the development of nT(reg) cells by limiting the extent of activation of the ERK and c-Rel signaling pathways.

The ability of Sos1 to oligomerize the adaptor protein LAT is separable from its guanine nucleotide exchange activity in vivo.

The activation of the small guanosine triphosphatase Ras by the guanine nucleotide exchange factor (GEF) Sos1 (Son of Sevenless 1) is a central feature of many receptor-stimulated signaling pathways. In developing T cells (thymocytes), Sos1-dependent activation of extracellular signal-regulated kinase (ERK) is required to stimulate cellular proliferation and differentiation. We showed that in addition to its GEF activity, Sos1 acted as a scaffold to nucleate oligomerization of the T cell adaptor protein LAT (linker for activation of T cells) in vivo. The scaffold function of Sos1 depended on its ability to bind to the adaptor protein Grb2. Furthermore, the GEF activity of Sos1 and the Sos1-dependent oligomerization of LAT were separable functions in vivo. Whereas the GEF activity of Sos1 was required for optimal ERK phosphorylation in response to T cell receptor (TCR) stimulation, the Sos1-dependent oligomerization of LAT was required for maximal TCR-dependent phosphorylation and activation of phospholipase C-γ1 and Ca(2+) signaling. Finally, both of these Sos1 functions were required for early thymocyte proliferation. Whereas transgenic restoration of either the GEF activity or the LAT oligomerization functions of Sos1 alone failed to rescue thymocyte development in Sos1-deficient mice, simultaneous reconstitution of these two signals in the same cell restored normal T cell development. This ability of Sos1 to act both as a RasGEF and as a scaffold to nucleate Grb2-dependent adaptor oligomerization may also occur in other Grb2-dependent pathways, such as those activated by growth factor receptors.

miRNA signature of mouse helper T cell hyper-proliferation.

Helper T cells from a mutant mouse model, LAT Y136F, hyper-proliferate and cause a severe lymphoproliferative disease that kills the mice by six months of age. LAT Y136F mice carry a tyrosine to phenylalanine mutation in the Linker for Activation of T cells (LAT) gene. This mutation leads to a number of changes in T cells that result in altered cytokine production including increased IL-4 production, increased proliferation, and decreased apoptosis. Hyper-proliferation of the mutant T cells contributes to lymphadenopathy, splenomegaly, and multi-organ T cell infiltration. miRNAs are short non-coding RNAs that regulate expression of cohorts of genes. This study investigates which miRNAs are expressed in LAT Y136F T cells and compares these to miRNAs expressed in wild type T cells that are undergoing proliferation in two other settings. The first setting is homeostatic proliferation, which was modeled by adoptive transfer of wild type T cells into T cell-deficient mice. The second setting is proliferation in response to infection, which was modeled by infection of wild type mice with the nematode H. polygyrus. By comparing miRNA expression in these three proliferative states (LAT Y136F hyper-proliferation, homeostatic proliferation and proliferation in response to H. polygyrus infection) to expression in wild type naïve CD4(+) T cells, we found miRNAs that were highly regulated in all three proliferative states (miR-21 and miR-146a) and some that were more specific to individual settings of proliferation such as those more specific for LAT Y136F lymphoproliferative disease (miR-669f, miR-155 and miR-466a/b). Future experiments that modulate levels of the miRNAs identified in this study may reveal the roles of these miRNAs in T cell proliferation and/or lymphoproliferative disease.

A phospholipase C-γ1-independent, RasGRP1-ERK-dependent pathway drives lymphoproliferative disease in linker for activation of T cells-Y136F mutant mice.

Mice expressing a germline mutation in the phospholipase C-γ1-binding site of linker for activation of T cells (LAT) show progressive lymphoproliferation and ultimately die at 4-6 mo age. The hyperactivated T cells in these mice show defective TCR-induced calcium flux but enhanced Ras/ERK activation, which is critical for disease progression. Despite the loss of LAT-dependent phospholipase C-γ1 binding and activation, genetic analysis revealed RasGRP1, and not Sos1 or Sos2, to be the major Ras guanine exchange factor responsible for ERK activation and the lymphoproliferative phenotype in these mice. Analysis of isolated CD4(+) T cells from LAT-Y136F mice showed altered proximal TCR-dependent kinase signaling, which activated a Zap70- and LAT-independent pathway. Moreover, LAT-Y136F T cells showed ERK activation that was dependent on Lck and/or Fyn, protein kinase C-θ, and RasGRP1. These data demonstrate a novel route to Ras activation in vivo in a pathological setting.

LAT-independent Erk activation via Bam32-PLC-γ1-Pak1 complexes: GTPase-independent Pak1 activation.

In T cells, the adaptor Bam32 is coupled to Erk activation downstream of the TCR by an unknown mechanism. We characterized in Jurkat cells and primary T lymphocytes a pathway dependent on Bam32-PLC-γ1-Pak1 complexes, in which Pak1 kinase activates Raf-1 and Mek-1, both upstream of Erk. In the Bam32-PLC-γ1-Pak1 complex, catalytically inactive PLC-γ1 is used as a scaffold linking Bam32 to Pak1. PLC-γ1(C-SH2) directly binds S141 of Bam32, preventing LAT-mediated activation of Ras by PLC-γ1. The Bam32-PLC-γ1 interaction enhances the binding of the SH3 domain of the phospholipase with Pak1. The PLC-γ1(SH3)-Pak1 interaction activates Pak1 independently of the small GTPases Rac1/Cdc42, previously described as being the only activators of Pak1 in T cells. Direct binding of the SH3 domain of PLC-γ1 to Pak1 dissociates inactive Pak1 homodimers, a mechanism required for Pak1 activation. We have thus uncovered a LAT/Ras-independent, Bam32-nucleated pathway that activates Erk signaling in T cells.

Deconstructing Ras signaling in the thymus.

Thymocytes must transit at least two distinct developmental checkpoints, governed by signals that emanate from either the pre-T cell receptor (pre-TCR) or the TCR to the small G protein Ras before emerging as functional T lymphocytes. Recent studies have shown a role for the Ras guanine exchange factor (RasGEF) Sos1 at the pre-TCR checkpoint. At the second checkpoint, the quality of signaling through the TCR is interrogated to ensure the production of an appropriate T cell repertoire. Although RasGRP1 is the only confirmed RasGEF required at the TCR checkpoint, current models suggest that the intensity and character of Ras activation, facilitated by both Sos and RasGRP1, will govern the boundary between survival (positive selection) and death (negative selection) at this stage. Using mouse models, we have assessed the independent and combined roles for the RasGEFs Sos1, Sos2, and RasGRP1 during thymocyte development. Although Sos1 was the dominant RasGEF at the pre-TCR checkpoint, combined Sos1/RasGRP1 deletion was required to effectively block development at this stage. Conversely, while RasGRP1 deletion efficiently blocked positive selection, combined RasGRP1/Sos1 deletion was required to block negative selection. This functional redundancy in RasGEFs during negative selection may act as a failsafe mechanism ensuring appropriate central tolerance.

Functional nanoscale organization of signaling molecules downstream of the T cell antigen receptor.

Receptor-regulated cellular signaling often is mediated by formation of transient, heterogeneous protein complexes of undefined structure. We used single and two-color photoactivated localization microscopy to study complexes downstream of the T cell antigen receptor (TCR) in single-molecule detail at the plasma membrane of intact T cells. The kinase ZAP-70 distributed completely with the TCRζ chain and both partially mixed with the adaptor LAT in activated cells, thus showing localized activation of LAT by TCR-coupled ZAP-70. In resting and activated cells, LAT primarily resided in nanoscale clusters as small as dimers whose formation depended on protein-protein and protein-lipid interactions. Surprisingly, the adaptor SLP-76 localized to the periphery of LAT clusters. This nanoscale structure depended on polymerized actin and its disruption affected TCR-dependent cell function. These results extend our understanding of the mechanism of T cell activation and the formation and organization of TCR-mediated signaling complexes, findings also relevant to other receptor systems.

Targeted Sos1 deletion reveals its critical role in early T-cell development.

Activation of the small G protein Ras is required for thymocyte differentiation. In thymocytes, Ras is activated by the Ras guanine exchange factors (RasGEFs) Sos1, Sos2, and RasGRP1. We report the development of a floxed allele of sos1 to assess the role of Sos1 during thymocyte development. Sos1 was required for pre-T-cell receptor (pre-TCR)- but not TCR-stimulated developmental signals. Sos1 deletion led to a partial block at the DN-to-DP transition. Sos1-deficient thymocytes showed reduced pre-TCR-stimulated proliferation, differentiation, and ERK phosphorylation. In contrast, TCR-stimulated positive selection, and negative selection under strong stimulatory conditions, remained intact in Sos1-deficient mice. Comparison of RasGEF expression at different developmental stages showed that relative to Sos2 and RasGRP1, Sos1 is most abundant in DN thymocytes, but least abundant in DP thymocytes. These data reveal that Sos1 is uniquely positioned to affect signal transduction early in thymocyte development.

The LAT story: a tale of cooperativity, coordination, and choreography.

The adapter molecule LAT is a nucleating site for multiprotein signaling complexes that are vital for the function and differentiation of T cells. Extensive investigation of LAT in multiple experimental systems has led to an integrated understanding of the formation, composition, regulation, dynamic movement, and function of LAT-nucleated signaling complexes. This review discusses interactions of signaling molecules that bind directly or indirectly to LAT and the role of cooperativity in stabilizing LAT-nucleated signaling complexes. In addition, it focuses on how imaging studies visualize signaling assemblies as signaling clusters and demonstrate their dynamic nature and cellular fate. Finally, this review explores the function of LAT based on the interpretation of mouse models using various LAT mutants.

Genetic evidence for the role of Erk activation in a lymphoproliferative disease of mice.

Germline mutation of the linker for activation of T cells (LAT) gene at the phospholipase C-gamma1 (PLC-gamma1)-binding site leads to a fatal lymphoproliferative disease in mice. The hyperactivated T cells that develop in these mice have defective T-cell antigen receptor (TCR)-induced calcium flux but enhanced mitogen-activated protein kinase (MAPK) activation. We used genetic analysis to investigate genes whose products might suppress MAPK activation and lymphoproliferative disease in LAT mutant mice. B-lymphocyte adaptor molecule of 32 kDa (Bam32) is a known mediator of MAPK activation in B cells. We recently reported that in CD4(+) T cells, Bam32 deficiency decreased MAPK activation and specifically extracellular-signal-regulated kinase (Erk) signaling, following TCR stimulation. By crossing the Bam32 null mutation onto the LAT knock-in background, we found that the Bam32 null mutation delayed the onset and decreased the severity of lymphoproliferative disease in LAT knock-in mice. The pulmonary lymphocyte infiltration seen in LAT knock-in mice was also markedly decreased in double-mutant mice. Additionally, Erk activation was diminished in LAT knock-in Bam32 knockout CD4(+) T cells. To more accurately determine the role of Erk in this delay of lymphoproliferative disease, we also bred a transgenic, hypersensitive Erk allele (the Erk2 sevenmaker mutant) onto the LAT knock-in Bam32 knockout double-mutant background. These triple transgenic mice demonstrated a role for Erk activation in lymphoproliferative disease caused by the LAT knock-in mutation.

Bam32: a novel mediator of Erk activation in T cells.

Bam32 (B lymphocyte adapter molecule of 32 kDa) is an adapter protein expressed in some hematopoietic cells including B and T lymphocytes. It was previously shown that Bam32-deficient mice have defects in various aspects of B cell activation including B cell receptor (BCR)-induced Erk activation, BCR-induced proliferation and T-independent antibody responses. In this study, we have examined the role of Bam32 in T cell activation using Bam32-deficient mice. By comparing CD4(+) T cells from lymph nodes of wild-type and Bam32-deficient mice, we found that Bam32 was required for optimal TCR-induced Erk activation, cytokine production, proliferation and actin-mediated spreading of CD4(+) T cells. These results indicate a novel pathway to Erk activation in T cells involving the adapter protein Bam32.

Dynamic movement of the calcium sensor STIM1 and the calcium channel Orai1 in activated T-cells: puncta and distal caps.

The proteins STIM1 and Orai1 are the long sought components of the store-operated channels required in T-cell activation. However, little is known about the interaction of these proteins in T-cells after engagement of the T-cell receptor. We found that T-cell receptor engagement caused STIM1 and Orai1 to colocalize in puncta near the site of stimulation and accumulate in a dense structure on the opposite side of the T-cell. FRET measurements showed a close interaction between STIM1 and Orai1 both in the puncta and in the dense cap-like structure. The formation of cap-like structures did not entail rearrangement of the entire endoplasmic reticulum. Cap formation depended on TCR engagement and tyrosine phosphorylation, but not on channel activity or Ca(2+) influx. These caps were very dynamic in T-cells activated by contact with superantigen pulsed B-cells and could move from the distal pole to an existing or a newly forming immunological synapse. One function of this cap may be to provide preassembled Ca(2+) channel components to existing and newly forming immunological synapses.

c-Cbl-mediated regulation of LAT-nucleated signaling complexes.

The engagement of the T-cell receptor (TCR) causes the rapid recruitment of multiple signaling molecules into clusters with the TCR. Upon receptor activation, the adapters LAT and SLP-76, visualized as chimeric proteins tagged with yellow fluorescent protein, transiently associate with and then rapidly dissociate from the TCR. Previously, we demonstrated that after recruitment into signaling clusters, SLP-76 is endocytosed in vesicles via a lipid raft-dependent pathway that requires the interaction of the endocytic machinery with ubiquitylated proteins. In this study, we focus on LAT and demonstrate that signaling clusters containing this adapter are internalized into distinct intracellular compartments and dissipate rapidly upon TCR activation. The internalization of LAT was inhibited in cells expressing versions of the ubiquitin ligase c-Cbl mutated in the RING domain and in T cells from mice lacking c-Cbl. Moreover, c-Cbl RING mutant forms suppressed LAT ubiquitylation and caused an increase in cellular LAT levels, as well as basal and TCR-induced levels of phosphorylated LAT. Collectively, these data indicate that following the rapid formation of signaling complexes upon TCR stimulation, c-Cbl activity is involved in the internalization and possible downregulation of a subset of activated signaling molecules.