Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb

Cancer immunotherapy is undergoing significant progress due to recent clinical successes by refined adoptive T-cell transfer and immunostimulatory monoclonal Ab (mAbs). B16F10-derived OVA-expressing mouse melanomas resist curative immunotherapy with either adoptive transfer of activated anti-OVA OT1 CTLs or agonist anti-CD137 (4-1BB) mAb. However, when acting in synergistic combination, these treatments consistently achieve tumor eradication. Tumor-infiltrating lymphocytes that accomplish tumor rejection exhibit enhanced effector functions in both transferred OT-1 and endogenous cytotoxic T lymphocytes (CTLs). This is consistent with higher levels of expression of eomesodermin in transferred and endogenous CTLs and with intravital live-cell two-photon microscopy evidence for more efficacious CTL-mediated tumor cell killing. Anti-CD137 mAb treatment resulted in prolonged intratumor persistence of the OT1 CTL-effector cells and improved function with focused and confined interaction kinetics of OT-1 CTL with target cells and increased apoptosis induction lasting up to six days postadoptive transfer. The synergy of adoptive T-cell therapy and agonist anti-CD137 mAb thus results from in vivo enhancement and sustainment of effector functions.Adoptive T-cell therapy is being developed following different approaches including infusion of expanded tumor infiltrating lymphocytes to preconditioned lympho-depleted hosts (1) and adoptive transfer of T cells genetically engineered to express tumor-specific T-cell receptors or chimeric antigen receptors (CARs) (2). The dazzling clinical success of CARs against leukemias (3, 4) is related to the fact that these chimeric receptors intracellularly include both signaling elements of the CD3-TCR (CD3ζ) and of costimulatory molecules (3). The intracellular costimulatory signaling domain with best reported effects so far is that of CD137 (4-1BB) (5).CD137 (4-1BB) is a TNFR family costimulatory receptor (TNFRSF9) that is expressed on activated T (6) and NK cells (7) and mediates costimulation of both types of lymphocytes (8). On CD8⁺ T cells ex vivo, CD137 ligation with the agonist antibodies determines increased proliferation, survival, memory formation and stronger effector functions in terms of both cytotoxicity and cytokine production (9). In vivo, anti-CD137 mAb protects adoptively transferred CTLs from activation-induced cell death resulting in better antitumor efficacy in a mouse myeloma model (10). Significant therapeutic effects against transplanted tumor models (11) have provided the rationale for currently ongoing phase I and II clinical trials ({"type":"clinical-trial","attrs":{"text":"NCT01471210","term_id":"NCT01471210"}}NCT01471210; {"type":"clinical-trial","attrs":{"text":"NCT01775631","term_id":"NCT01775631"}}NCT01775631; {"type":"clinical-trial","attrs":{"text":"NCT01307267","term_id":"NCT01307267"}}NCT01307267) (8).NK cells up-regulate CD137 and ligation by anti-CD137 mAb enhances NK-mediated antibody-dependent cellular cytotoxicity functions resulting in synergistic effects with anti-CD20 (12), anti-HER2 (13) and anti-EGFR (14) mAb. CD137 expression can also be induced on dendritic cells (15), tumor endothelial cells (16), B cells (17), and myeloid leukocytes (18) upon activation. Although positive effects of CD137 ligation for CD8⁺ T-cell memory generation are well explored (9, 19), its relevance for enhancing effector function in solid tumor lesions in vivo has not been established. In this study we show a synergy of adoptively transferred and endogenous CD8⁺ T cells against B16F10 melanoma that depends on the ability of both CTL populations to receive costimulation via CD137. Flow-cytometry of tumor-rejecting lymphocyte infiltrates and intravital microscopy of tumors provide evidence that anti-CD137 mAb therapy sustained the efficacy of more focused anti-tumor CTLs.     

CD81 costimulation skews CAR transduction toward naive T cells

Adoptive cellular therapy using chimeric antigen receptors (CARs) has revolutionized our treatment of relapsed B cell malignancies and is currently being integrated into standard therapy. The impact of selecting specific T cell subsets for CAR transduction remains under investigation. Previous studies demonstrated that effector T cells derived from naive, rather than central memory T cells mediate more potent antitumor effects. Here, we investigate a method to skew CAR transduction toward naive T cells without physical cell sorting. Viral-mediated CAR transduction requires ex vivo T cell activation, traditionally achieved using antibody-mediated strategies. CD81 is a T cell costimulatory molecule that when combined with CD3 and CD28 enhances naive T cell activation. We interrogate the effect of CD81 costimulation on resultant CAR transduction. We identify that upon CD81-mediated activation, naive T cells lose their identifying surface phenotype and switch to a memory phenotype. By prelabeling naive T cells and tracking them through T cell activation and CAR transduction, we document that CD81 costimulation enhanced naive T cell activation and resultantly generated a CAR T cell product enriched with naive-derived CAR T cells.

Genetic manipulation of T cells has enabled adoptive T cell therapy to be translated to the clinic (1–10). Chimeric antigen receptor (CAR) therapy has evoked recent enthusiasm upon mediating curative outcomes in aggressive, refractory B cell malignancies (1–7, 11–15), leading to Food and Drug Administration approval (16–18). The process of ex vivo transduction and expansion of T cells to express CARs influences the phenotype, function, and ultimate fate of the final CAR T cell product (19–23). Preclinical data in animal models indicate that selecting specific T cell subsets for CAR transduction improves efficacy (21, 22, 24–26). Naive-derived T cells have been shown to exhibit greater replicative capacity, persistence, and antitumor function, compared with both effector- and memory-derived T cells (19, 20, 27). Naive CD4⁺ T cells, specifically, have a critical role in enhancing the cytotoxic effect of the CD8⁺ cooperating central memory cell subset (21). Furthermore, the translational CAR experience demonstrates that the presence of cells consistent with the naive and early memory phenotype in premanufactured T cell products correlates with successful clinical responses in both pediatric and adult B cell leukemia (28–30). Here, we explore if selective activation of naive T cells can result in skewing of transduction toward this specific T cell subset without the need for physical subset sorting.CAR constructs rely on intrinsic costimulatory signals, such as the intracellular domains of CD28 or 41BB, for efficacy (1–9). Here we focus on exogenous costimulatory signals necessary to induce proliferation and permit viral-mediated gene transfer. Prior to CAR transduction and antigen encounter, the majority of T cells are in a state of rest. Resting T cells mandate primary and costimulatory signals for activation (31, 32). CD28 is best known for its ability to costimulate T cells (33–38) and along with CD3 activation renders T cells susceptible to viral transduction (1, 39). CD81 is a member of the tetraspanin family that physically associates with CD4 and CD8 on the surface of T cells. CD81 was shown to have independent costimulatory properties and, when used with anti-CD3 and -CD28 antibodies, preferentially activates naive T cells as compared with effector and memory T cells, despite conserved surface CD81 expression across T cell subsets (40). Tetraspanins have no known cell-surface ligands, and therefore antibodies are used to engage and stimulate them. CD81 is the only tetraspanin whose complete three-dimensional structure has been solved (41). Moreover, the crystal structure of 5A6, the anti-CD81 antibody used in our study, in complex with CD81 has also been most recently solved (42). These authors demonstrate that engagement by this antibody changes the conformation of the large extracellular loop of the CD81 molecule. This conformational change may affect the interaction of CD81 with its associated CD4 and CD8 molecules.Here, we costimulate purified T cells with CD81 as a proof of principle to illustrate that the in vitro activation window prior to CAR transduction can be leveraged to favor transduction of a specific T cell subset.     

CD8 coreceptor-mediated focusing can reorder the agonist hierarchy of peptide ligands recognized via the T cell receptor

CD8⁺ T cells are inherently cross-reactive and recognize numerous peptide antigens in the context of a given major histocompatibility complex class I (MHCI) molecule via the clonotypically expressed T cell receptor (TCR). The lineally expressed coreceptor CD8 interacts coordinately with MHCI at a distinct and largely invariant site to slow the TCR/peptide-MHCI (pMHCI) dissociation rate and enhance antigen sensitivity. However, this biological effect is not necessarily uniform, and theoretical models suggest that antigen sensitivity can be modulated in a differential manner by CD8. We used two intrinsically controlled systems to determine how the relationship between the TCR/pMHCI interaction and the pMHCI/CD8 interaction affects the functional sensitivity of antigen recognition. Our data show that modulation of the pMHCI/CD8 interaction can reorder the agonist hierarchy of peptide ligands across a spectrum of affinities for the TCR.

CD8⁺ T cells are critical for protective immunity against intracellular pathogens and various tumors. At the molecular level, activation is triggered by foreign or mutated peptide fragments presented on the cell surface by major histocompatibility complex class I (MHCI) molecules, which act as ligands for the somatically rearranged T cell receptor (TCR) and the germline-encoded coreceptor CD8 (1, 2). The clonotypically expressed TCR confers antigen specificity by interacting with the peptide-binding platform of MHCI, which comprises the α1 and α2 domains, whereas the lineally expressed coreceptor CD8 is known to enhance antigen sensitivity by interacting primarily with the α3 domain of MHCI (3–7). This latter interaction is biophysically and spatially independent of peptide-MHCI (pMHCI) engagement via the TCR (8). However, the largely invariant nature of the pMHCI/CD8 interaction does not necessarily translate into a uniform gain of function, and theoretical studies have suggested that antigen sensitivity can be modulated in a differential manner, potentially altering the agonist hierarchy of peptide ligands for any given TCR (9–11).The pMHCI/CD8 interaction slows the dissociation rate of the TCR/pMHCI interaction (9, 12). Functional sensitivity depends nonmonotonically on this dissociation rate (13), as long as the system is limited by MHCI (10, 14, 15). The nature of this relationship implies that functional sensitivity reaches a maximum at a particular dissociation rate. Strong agonists are relatively insensitive to modulation of the dissociation rate, because the curve has a negligible slope in the vicinity of the optimal value. In contrast, weak agonists are typically characterized by faster dissociation rates, modulation of which markedly alters functional sensitivity (16). Accordingly, the pMHCI/CD8 interaction generally acts to increase agonist potency, maximizing the number of peptide ligands that can be recognized via a given TCR. However, theoretical models predict that ligands with dissociation rates below or close to the optimal value will respond differently, amounting to a differential focusing effect, whereby strong agonists can become less potent at dissociation rates beyond the optimal value. If operative in vivo, such an effect could allow individual clonotypes to focus on salient ligands (9), reconciling the inherent need for cross-reactivity with the inherent need for specificity (17).We used two monoclonal systems incorporating biophysically defined peptide ligands and variants of MHCI with altered coreceptor-binding properties to test the differential focusing hypothesis experimentally. In line with earlier predictions, we found that modulation of the pMHCI/CD8 interaction reordered the agonist hierarchy of peptide ligands recognized via the TCR.     

From the Cover: Anti-CD47 antibody–mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response

Mobilization of the T-cell response against cancer has the potential to achieve long-lasting cures. However, it is not known how to harness antigen-presenting cells optimally to achieve an effective antitumor T-cell response. In this study, we show that anti-CD47 antibody–mediated phagocytosis of cancer by macrophages can initiate an antitumor T-cell immune response. Using the ovalbumin model antigen system, anti-CD47 antibody–mediated phagocytosis of cancer cells by macrophages resulted in increased priming of OT-I T cells [cluster of differentiation 8-positive (CD8⁺)] but decreased priming of OT-II T cells (CD4⁺). The CD4⁺ T-cell response was characterized by a reduction in forkhead box P3-positive (Foxp3⁺) regulatory T cells. Macrophages following anti-CD47–mediated phagocytosis primed CD8⁺ T cells to exhibit cytotoxic function in vivo. This response protected animals from tumor challenge. We conclude that anti-CD47 antibody treatment not only enables macrophage phagocytosis of cancer but also can initiate an antitumor cytotoxic T-cell immune response.Antigen presentation is the process by which innate immune cells such as macrophages and dendritic cells (antigen-presenting cells, APC) acquire antigens and present them to T cells to initiate the adaptive immune response. How APCs shape the immune response by both degrading antigens and preserving antigens for presentation to T cells has been a longstanding area of interest (1). Recently, the mechanism of antigen recognition by APCs has been shown to affect the preference of MHC I versus MHC II antigen-presentation pathways. For instance, mannose receptor-mediated endocytosis on dendritic cells has been associated with MHC I antigen presentation, whereas scavenger receptor-mediated endocytosis has been associated with MHC II presentation (2). Moreover, the functional outcomes of antigen presentation have been shown to be context dependent. For instance, targeting antigens to DEC-205 using monoclonal antibodies induced tolerance under noninflammatory conditions but mediated immunogenicity under activating conditions by cluster of differentiation 40 ligand (CD40L) (3). Harnessing APCs to enhance the antitumor T-cell response offers an exciting strategy for cancer immunotherapy. The ability of the T-cell immune response to be mobilized successfully against cancer has been demonstrated through preclinical and clinical studies of anti-CTLA4 antibody for T-cell activation (4).Phagocytosis by macrophages relies on the cell’s recognition of prophagocytic (“eat me”) and antiphagocytic (“don’t eat me”) signals on target cells. Anti-CD47 blocking monoclonal antibodies (mAbs) induce macrophage phagocytosis of cancer cells by inhibiting an important antiphagocytic signal, allowing prophagocytic signals to dominate (5, 6). CD47 is highly expressed on cancer cells as compared with normal cells (5, 6) and interacts with the ligand signal regulatory protein α (SIRP-α) on macrophages (7). This interaction results in phosphorylation of immunoreceptor tyrosine-based inhibition (ITIM) motifs on SIRP-α’s cytoplasmic tail and the recruitment of Src homology phosphatase-1 (SHP-1) and SHP-2 phosphatases, which is thought to block phagocytosis by preventing myosin-IIA accumulation at the phagocytic synapse (8–12). We have demonstrated the therapeutic efficacy of anti-CD47 blocking mAbs against xenograft human cancers growing in immunodeficient mice, including cancers such as leukemia (5, 13), lymphoma (14), and multiple myeloma (15), solid tumors, including breast, colon, prostate, and bladder cancers, and sarcomas (6, 16). Whether the adaptive immune response also can be recruited against the cancer after anti-CD47 mAb treatment has not been tested, because the immunodeficient mice used to establish the xenograft models lack T, B, and NK cells. In this study, we tested the hypothesis that anti-CD47 antibody–mediated phagocytosis of cancer cells can facilitate an antitumor T-cell immune response.     

From the Cover: 90Y-daclizumab,an anti-CD25 monoclonal antibody,provided responses in 50% of patients with relapsed Hodgkin’s lymphoma

Despite significant advances in the treatment of Hodgkin’s lymphoma (HL), a significant proportion of patients will not respond or will subsequently relapse. We identified CD25, the IL-2 receptor alpha subunit, as a favorable target for systemic radioimmunotherapy of HL. The scientific basis for the clinical trial was that, although most normal cells with exception of Treg cells do not express CD25, it is expressed by a minority of Reed–Sternberg cells and by most polyclonal T cells rosetting around Reed–Sternberg cells. Forty-six patients with refractory and relapsed HL were evaluated with up to seven i.v. infusions of the radiolabeled anti-CD25 antibody ⁹⁰Y-daclizumab. ⁹⁰Y provides strong β emissions that kill tumor cells at a distance by a crossfire effect. In 46 evaluable HL patients treated with ⁹⁰Y-daclizumab there were 14 complete responses and nine partial responses; 14 patients had stable disease, and nine progressed. Responses were observed both in patients whose Reed–Sternberg cells expressed CD25 and in those whose neoplastic cells were CD25⁻ provided that associated rosetting T cells expressed CD25. As assessed using phosphorylated H2AX (γ-H2AX) as a bioindicator of the effects of radiation exposure, predominantly nonmalignant cells in the tumor microenvironment manifested DNA damage, as reflected by increased expression of γ-H2AX. Toxicities were transient bone-marrow suppression and myelodysplastic syndrome in six patients who had not been evaluated with bone-marrow karyotype analyses before therapy. In conclusion, repeated ⁹⁰Y-daclizumab infusions directed predominantly toward nonmalignant T cells rosetting around Reed–Sternberg cells provided meaningful therapy for select HL patients.Treatment with combination chemotherapy, radiation, and hematopoietic stem cell transplantation has increased the disease-free survival in Hodgkin’s lymphoma (HL) from less than 5% in 1963 to more than 80% at present (1–6). Recently the US Food and Drug Administration approved brentuximab vedotin for the treatment of relapsed HL (7). Furthermore the anti-PD1 agent pembrolizumab has shown promising results in classic HL (8). Nevertheless, a significant fraction of patients do not respond to treatment or subsequently relapse. To date more than 30 different mAb preparations directed toward antigens expressed by malignant Reed–Sternberg cells have been studied (6). These include mAbs linked to drugs or toxins targeting CD25 or CD30 expressed on Reed–Sternberg cells (6–11). Brentuximab vedotin, an anti-CD30 antibody drug conjugate, has induced a significant number of responses in refractory HL (7, 11). Although other antibody immunotoxins have demonstrated some clinical efficacy, they have yielded few complete responses (CRs) (6, 9, 10). An alternative strategy has been to arm mAbs with radionuclides. Radioimmunotherapy using ⁹⁰Y–anti-ferritin and ¹³¹I–anti-CD30 antibodies has resulted in partial (PRs) and CRs in HL (12–15). Deficiencies with these approaches reflect the lack of tumor specificity of ferritin-targeted antibodies and the small number of CD30-expressing Reed–Sternberg cells in the tumor.As an alternative, we identified CD25, the IL-2 receptor alpha subunit (IL-2Rα), as a more favorable target for systemic radioimmunotherapy of HL (16–22). The scientific rationale is that, with the exception of Treg cells, CD25 is not expressed by normal resting lymphoid cells, but it is expressed on both a minority of Reed–Sternberg cells and, critically, on T cells rosetting around Reed–Sternberg cells in HL (6, 23, 24). ⁹⁰Y, an energetic β particle emitter with a mean tissue path length of 5 mm and a maximal path length of 11 mm, acts through “crossfire” throughout tumor masses, providing a strategy for killing tumor cells at a distance of several cell diameters, including Reed–Sternberg cells that lack CD25 expression provided that T cells in their vicinity express the target antigen (16, 23, 24). In the current phase II trial we treated 46 patients with recurrent or refractory HL with ⁹⁰Y-daclizumab every 6–10 wk for up to seven doses, depending on hematological recovery. The activity of ⁹⁰Y used in the present trial was determined on the basis of three previous phase I/II dose-escalation trials of ⁹⁰Y–anti-CD25 performed in patients with lymphoproliferative disorders (16).     

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR–CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR–CD3 complexes. We found that only TCR–CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR–pMHC affinity determined the density of TCR–CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR–CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.The activation of T cells orchestrates an adaptive immune response by translating antigen binding to the T-cell receptor (TCR) into appropriate cellular responses (1–4). The αβ TCR engages MHC molecules (or HLA) bound to antigenic peptides (pMHC) on the surface of antigen-presenting cells (5). The interaction of the TCR with pMHC is highly specific because T cells are able to distinguish rare foreign pMHC among abundant self pMHC molecules (6). TCR signaling is also extremely sensitive; even a single pMHC molecule is sufficient to trigger activation (7–9). TCRs are noncovalently coupled to the conserved multisubunit CD3 complex, comprising CD3εγ, CD3εδ, and CD3ζζ dimers (10), whose immunoreceptor tyrosine-based activation motifs (ITAMs) are phosphorylated upon pMHC engagement by the nonreceptor tyrosine kinase Lck (1, 2). ITAM phosphorylation is required for the recruitment and phosphorylation of the ζ-chain-associated protein kinase 70 kDa (Zap70) and the adaptor linker for activation of T cells (Lat) (11) to mediate downstream activation responses (12). Phosphorylation of the TCR–CD3 complex is one of the earliest detectable biochemical events in T-cell signaling and already at this level, important “activation decisions” are being made. For example, when the extent of ITAM phosphorylation was modulated through specific mutations, low levels of TCR–CD3 phosphorylation were sufficient for signaling through the Zap70–SLP-76–Lat pathway and cytokine production, whereas high levels of TCR–CD3 phosphorylation were required for Vav1-Numb-Notch signaling and T-cell proliferation (12–14). However, how the TCR–CD3 complex encodes both the quality and quantity of pMHC molecules and steers signaling activities toward appropriate cellular outcomes is not fully understood (1–4).Although many of the molecular players and TCR signaling pathways have been identified and characterized by biochemical and genetic approaches (12, 15), the precise mechanism by which the binding of the TCR to pMHC results in phosphorylation of the TCR–CD3 complex, referred to as TCR triggering, still remains contested (1, 16). There is increasing evidence that the spatial reorganization of the TCR into micrometer- and submicron-sized clusters is involved in regulating T-cell activation (2, 11, 17–19). With the advent of superresolution fluorescence microscopy, we have gained a much more nuanced picture of the spatial organization of TCR signaling proteins (3, 20). In particular, single-molecule localization microscopy [SMLM, including photoactivated localization microscopy (PALM) (21) and direct stochastic optical reconstruction microscopy (dSTORM) (22)] was used to report that at least a proportion of TCRs were organized into small clusters that were 30–300 nm in diameter, termed “nanoclusters” (23, 24). Similarly, Lat (23–25), Lck (26), and Zap70 (24, 27) were also found to reside in nanoclusters that are extensively remodeled during T-cell activation. The link between preexisting and pMHC-induced nanoclustering and signaling activities is not clear at present and is the focus of the present study.To identify the functional role of TCR nanoclusters, we used two-color SMLM data and integrated a cluster detection method, density-based spatial clustering of applications with noise (DBSCAN) (28) with a customized colocalization analysis (29). This process allowed us to distinguish phosphorylated from nonphosphorylated TCR–CD3 complex clusters in intact T cells and identify the spatial organization at which individual TCR–CD3 complexes had the highest signaling efficiency. We found that not all TCR–CD3 complexes had the same likelihood of being phosphorylated, even with excess doses of high-affinity pMHC molecules. The signaling efficiency of the TCR–CD3 complex was dependent upon the distance to neighboring complexes so that dense nanoclusters had the highest TCR triggering efficiency.     

Structural basis for substrate specificity of heteromeric transporters of neutral amino acids

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo–electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.

Amino acids play a central role in cellular metabolism. Dysregulation of both intra- and extracellular amino acid concentrations is associated with pathological conditions (1). Amino acid transfer across the plasma membrane is mediated by specific transporters that bind and transport these molecules from the extracellular medium into the cell or vice versa.Heteromeric amino acid transporters (HATs) are a family of amino acid transporters comprised by a heavy subunit and a light subunit, linked by a conserved disulfide bridge (2). Heavy subunits (SLC3 family) are ancillary proteins required for trafficking the holotransporter to the plasma membrane (2), whereas the light subunits (LATs; SLC7 family) transport amino acids and confer substrate specificity to the heterodimer (2). HATs are amino acid exchangers that harmonize amino acid concentrations at each side of the plasma membrane and as such they play a critical role in amino acid homeostasis (1, 3).The physiological relevance of HATs is highlighted by their role in cancer and several inherited diseases (4–8). HAT neutral amino acid transporters in particular are gaining momentum as several mutations linked to human diseases have recently been identified, and new physiological roles for this group of transporters have been uncovered using knockout mouse models (8–13). Several loss-of-function mutations in human LAT2/CD98hc (SLC7A8/SLC3A2) are associated with age-related hearing loss (ARHL) (9) and cataracts (10). Also, some coding variants are linked to an increased risk of autism spectrum disorder (14). In addition, hLAT2/CD98hc overexpression in pancreatic cancer cells sustains glutamine-dependent mTOR activation to promote glycolysis and chemoresistance (15). This observation thus points to hLAT2/CD98hc as a potential pharmacological target in this particular type of cancer. On the other hand, LAT1/CD98hc (SLC7A5/SLC3A2), which is also linked to cancer (4, 7), participates in brain development and autism spectrum disorder (12). Finally, Asc1/CD98hc (SLC7A10/SLC3A2) is considered a target to regulate glutamatergic neurotransmission in some cognitive disorders, such as schizophrenia (16, 17), and a relevant player in adipocyte lipid storage, obesity, and insulin resistance (18).Several atomic structures of HATs (19–24) and LATs (25) have recently been described, thus paving the way for the dissection of the molecular transport mechanisms. The substrate-binding site of LATs determined in complex with a substrate or competitive inhibitors shows a conserved design consisting of two unwound segments of transmembrane (TM) 1 and TM6, which contain residues that recognize the α-amino and carboxyl groups of the substrate (21–25). Each member of the HAT family displays a preference for transporting a certain set of substrates (2). LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc transport neutral amino acids but of different sizes. LAT1 is specialized in large neutral amino acids but it is inefficient for L-glutamine, and it does not transport small amino acids. LAT2 transports both large and small neutral amino acids, and it is highly efficient for L-glutamine. Finally, Asc1 mediates the preferential uptake of small neutral amino acids, including D-isomers, particularly D-serine (26–28).Despite recent advances in resolving the structure of several HATs, the molecular mechanisms explaining why each member of the family shows exquisite preference for certain substrates but not others are mostly unknown. Here, we addressed the structural bases of substrate specificity in the HAT family. To this end, we used cryo–electron microscopy (cryo-EM) to determine the structure of human LAT2/CD98hc in inward-facing open and apo conformation. We used this structure to study substrate-binding determinants by combining Protein Energy Landscape Exploration (PELE) and molecular dynamics (MD), together with mutational and functional studies. We reveal that a few residues present in the substrate-binding pocket and nearby regions determine substrate preference, and we demonstrate how the substrate preference of several HATs can be interconverted. In addition, a region located at a certain distance of the substrate cavity but whose structure critically influences the conformation of the substrate-binding site also regulates substrate preference. This region accumulates mutations associated with ARHL and cataracts that alter hLAT2 substrate specificity.Our work uncovers key structural determinants that govern, by different mechanisms, the differences in substrate specificity found within HAT members of neutral amino acid transporters. It also provides the structural bases for mutations in LAT2/CD98hc associated with deafness and cataracts.     

Pro-inflammatory T helper 17 directly harms oligodendrocytes in neuroinflammation

T helper (Th)17 cells are considered to contribute to inflammatory mechanisms in diseases such as multiple sclerosis (MS). However, the discussion persists regarding their true role in patients. Here, we visualized central nervous system (CNS) inflammatory processes in models of MS live in vivo and in MS brains and discovered that CNS-infiltrating Th17 cells form prolonged stable contact with oligodendrocytes. Strikingly, compared to Th2 cells, direct contact with Th17 worsened experimental demyelination, caused damage to human oligodendrocyte processes, and increased cell death. Importantly, we found that in comparison to Th2 cells, both human and murine Th17 cells express higher levels of the integrin CD29, which is linked to glutamate release pathways. Of note, contact of human Th17 cells with oligodendrocytes triggered release of glutamate, which induced cell stress and changes in biosynthesis of cholesterol and lipids, as revealed by single-cell RNA-sequencing analysis. Finally, exposure to glutamate decreased myelination, whereas blockade of CD29 preserved oligodendrocyte processes from Th17-mediated injury. Our data provide evidence for the direct and deleterious attack of Th17 cells on the myelin compartment and show the potential for therapeutic opportunities in MS.

Multiple sclerosis (MS) is a disabling inflammatory disease of the central nervous system (CNS) characterized by demyelination as a key pathological hallmark (1). Loss of metabolic support and axonal myelination provided by oligodendrocytes not only impairs neurotransmission but also compromises neuronal homeostasis, leading to neuroaxonal vulnerability (2, 3). Chronic demyelination is therefore considered to play a major role in the progression of neurological disability in MS (4–6). The pharmacological protection of oligodendrocytes has been discussed as a new therapeutic strategy for MS (7) and is especially appealing in light of recent results showing that oligodendrocyte damage is partially reversible (8). However, the mechanisms underlying immune-mediated oligodendrocyte injury in neuroinflammation are still only partially understood.Proinflammatory CD4⁺ T cells are considered to play a major role in neuroinflammatory processes in MS and in its animal model, experimental autoimmune encephalomyelitis (EAE) (9–11). In particular, interleukin (IL)-23–polarized T helper (Th)17 cells coexpressing T-bet and ROR-γ are considered pathogenic in both EAE (12, 13) and in MS (13–15). Interestingly, in MS subjects, reduced Th17 responses after hematopoietic stem cell transplantation (16) or the exclusion of the double-positive Th17/1 cells from the CNS compartment after treatment with natalizumab (17) is associated with a dramatic decrease in the accumulation or expansion of demyelinating lesions, while interfering with IL-17A-cytokine signaling had moderate effects on disease severity (18). The capacity of IL-23–skewed Th17 cells to destabilize the blood–brain barrier (BBB) and recruit antigen-presenting cells to the CNS compartment in neuroinflammation is well established (13, 19, 20), but it is not clear whether Th17 cells cause tissue injury themselves. Previous studies have demonstrated that activated CD4⁺ T cells can exert cytotoxicity toward human and rodent oligodendrocytes (21–23) in vitro and impair remyelination in a toxic mouse model (24). Of note, older in vitro reports, from prior to the discovery of Th17 cells, using human oligodendrocytic cell lines rather excluded a soluble cytokine mediator of adult oligodendrocyte lysis (21, 25). Therefore, our goal was to unravel the direct influence of Th17 cells on the myelin compartment in neuroinflammation.     

From the Cover: Functional evidence for TCR-intrinsic specificity for MHCII

How T cells become restricted to binding antigenic peptides within class I or class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) via clonotypic T-cell receptors (TCRs) remains debated. During development, if TCR–pMHC interactions exceed an affinity threshold, a signal is generated that positively selects the thymocyte to become a mature CD4⁺ or CD8⁺ T cell that can recognize foreign peptides within MHCII or MHCI, respectively. But whether TCRs possess an intrinsic, subthreshold specificity for MHC that facilitates sampling of the peptides within MHC during positive selection or T-cell activation is undefined. Here we asked if increasing the frequency of lymphocyte-specific protein tyrosine kinase (Lck)-associated CD4 molecules in T-cell hybridomas would allow for the detection of subthreshold TCR–MHC interactions. The reactivity of 10 distinct TCRs was assessed in response to selecting and nonselecting MHCII bearing cognate, null, or “shaved” peptides with alanine substitutions at known TCR contact residues: Three of the TCRs were selected on MHCII and have defined peptide specificity, two were selected on MHCI and have a known pMHC specificity, and five were generated in vitro without defined selecting or cognate pMHC. Our central finding is that IL-2 was made when each TCR interacted with selecting or nonselecting MHCII presenting shaved peptides. These responses were abrogated by anti-CD4 antibodies and mutagenesis of CD4. They were also inhibited by anti-MHC antibodies that block TCR–MHCII interactions. We interpret these data as functional evidence for TCR-intrinsic specificity for MHCII.Positive and negative selection limit the αβT-cell repertoire to cells expressing clonotypic T-cell receptors (TCRs) that distinguish the antigenicity of peptides embedded within class I and class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) based on their source of origin (i.e., self or foreign) (1–4). Approximately 7.5% of CD4⁺CD8⁺ double-positive (DP) thymocytes express TCRs that interact with self-pMHC above an affinity threshold required for positive selection, whereas 7.5% cross a higher affinity threshold that mediates negative selection and the remaining TCRs fail to direct positive selection (5). The rules that restrict TCR recognition of antigenic peptides within MHCI or MHCII are unresolved.Two models have been proposed to explain MHC restriction. One posits that restriction is imposed by CD4 or CD8 during thymocyte development to eliminate TCRs that recognize non-MHC ligands (2, 6). Here, the CD4- and CD8-associated Src kinase, p56^Lck [lymphocyte-specific protein tyrosine kinase (Lck)], is sequestered away from the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR-associated CD3δε, CD3γε, and CD3ζζ signaling modules. Positively selecting signals are then generated in thymocytes expressing TCRs that bind MHCII or MHCI together with CD4 or CD8, respectively, as this localizes Lck to the ITAMs. Those thymocytes expressing TCRs that do not bind MHCI or MHCII would fail to localize Lck to the ITAMs and die. In the second model, germ line-encoded complementary determining regions (CDR) 1 and 2 allow each clonotypic TCR to bind distinct classes and alleles of MHC molecules via unique yet specific recognition codons that impose a canonical docking polarity and MHC restriction (1, 3, 4, 7, 8). Although it is not obvious that these models are mutually exclusive, the key distinction is that in the first model the randomly generated preselection TCR repertoire would contain TCRs that do and do not bind pMHC, whereas in the second model most if not all TCRs would have a specificity for MHC that is germ line-encoded, regardless of the class or allele of MHC.The canonical docking polarity of TCRs on MHCI or MHCII observed in crystal structures, and the CDR1 and CDR2 contacts therein, provides evidence for germ line-encoded TCR–MHC interactions for positively selected TCRs (1, 3, 4, 7, 8). But this is taken as supporting either model, as germ line-encoded contacts are likely to be required to allow the formation of a TCR–CD3–pMHC–CD4/CD8 macrocomplex that situates the CD3 ITAMs and Lck in a functionally mandated orientation (1–4, 6, 9, 10). Structural insights from positively selected TCRs thus do not allow the basis of MHC restriction to be cleanly addressed, and functional data that support either model have been reported (11–15).An open question that can shed light on the similarities and differences between the two models is whether TCRs participate in subthreshold scanning of MHC (4, 16). Scanning would allow a TCR to dock on MHC and survey its contents for peptides that increase the duration of TCR–pMHC interactions, via contacts with clonotypic CDR3s, and allow the formation of a TCR–CD3–pMHC–CD4/CD8 macrocomplex that generates signals (4, 10). In the co-receptor imposed model, a diverse preselection repertoire would contain TCRs with no intrinsic capacity to bind MHC, TCRs that interact with pMHC by atypical modalities, and TCRs that interact with a composite pMHC surface in a canonical modality in a lock-and-key manner akin to antibody–antigen recognition (2, 6). Once selected, this last group of TCRs would be predicted to scan composite pMHC with shapes (i.e., topology and chemical characteristics) related to the selecting pMHC—presumably the same MHC, or similar allelic variant, presenting related peptides. In the germ line-encoded recognition model, TCR scanning of MHC via recognition codons would be intrinsic to most if not all TCRs, regardless of the class of MHC, allelic variants, or the peptide sequence therein (4). At present, functional evidence for TCR scanning of MHC is lacking, regardless of whether it is MHC class-, allele-, and peptide sequence-dependent.Recently, the frequency of Lck-associated CD4 molecules was proposed to influence if a TCR–pMHC interaction is of sufficient duration to direct a specific cell fate decision, such as negative selection (17). We thus hypothesized that genetically increasing the frequency of CD4–Lck association should allow for the detection of subthreshold TCR–pMHC interactions that are normally of insufficient duration to elicit a functional response. Here we show that T-cell hybridomas expressing 10 distinct TCRs along with a CD4–Lck fusion make IL-2 in response to APCs expressing selecting or nonselecting MHCII, regardless of the sequence of the presented peptide. These responses were independent of positive selection on MHCII, as TCRs that were positively selected on MHCI, or generated in vitro and thus not thymically selected, yielded similar responses. These data provide functional evidence for subthreshold TCR scanning of MHCII that is independent of the class of MHC, the allele, or the peptide sequence therein.     

Molecular architecture of the αβ T cell receptor–CD3 complex

αβ T-cell receptor (TCR) activation plays a crucial role for T-cell function. However, the TCR itself does not possess signaling domains. Instead, the TCR is noncovalently coupled to a conserved multisubunit signaling apparatus, the CD3 complex, that comprises the CD3εγ, CD3εδ, and CD3ζζ dimers. How antigen ligation by the TCR triggers CD3 activation and what structural role the CD3 extracellular domains (ECDs) play in the assembled TCR–CD3 complex remain unclear. Here, we use two complementary structural approaches to gain insight into the overall organization of the TCR–CD3 complex. Small-angle X-ray scattering of the soluble TCR–CD3εδ complex reveals the CD3εδ ECDs to sit underneath the TCR α-chain. The observed arrangement is consistent with EM images of the entire TCR–CD3 integral membrane complex, in which the CD3εδ and CD3εγ subunits were situated underneath the TCR α-chain and TCR β-chain, respectively. Interestingly, the TCR–CD3 transmembrane complex bound to peptide–MHC is a dimer in which two TCRs project outward from a central core composed of the CD3 ECDs and the TCR and CD3 transmembrane domains. This arrangement suggests a potential ligand-dependent dimerization mechanism for TCR signaling. Collectively, our data advance our understanding of the molecular organization of the TCR–CD3 complex, and provides a conceptual framework for the TCR activation mechanism.T cells are key mediators of the adaptive immune response. Each αβ T cell contains a unique αβ T-cell receptor (TCR), which binds antigens (Ags) displayed by major histocompatibility complexes (MHCs) and MHC-like molecules (1). The TCR serves as a remarkably sensitive driver of cellular function: although TCR ligands typically bind quite weakly (1–200 μM), even a handful of TCR ligands are sufficient to fully activate a T cell (2, 3). The TCR does not possess intracellular signaling domains, uncoupling Ag recognition from T-cell signaling. The TCR is instead noncovalently associated with a multisubunit signaling apparatus, consisting of the CD3εγ and CD3εδ heterodimers and the CD3ζζ homodimer, which collectively form the TCR–CD3 complex (4, 5). The CD3γ/δ/ε subunits each consist of a single extracellular Ig domain and a single immunoreceptor tyrosine-based activation motif (ITAM), whereas CD3ζ has a short extracellular domain (ECD) and three ITAMs (6–11). The TCR–CD3 complex exists in 1:1:1:1 stoichiometry for the αβTCR:CD3εγ:CD3εδ:CD3ζζ dimers (12). Phosphorylation of the intracellular CD3 ITAMs and recruitment of the adaptor Nck lead to T-cell activation, proliferation, and survival (13, 14). Understanding the underlying principles of TCR–CD3 architecture and T-cell signaling is of therapeutic interest. For example, TCR–CD3 is the target of therapeutic antibodies such as the immunosuppressant OKT3 (15), and there is increasing interest in manipulating T cells in an Ag-dependent manner by using naturally occurring and engineered TCRs (16).Assembly of the TCR–CD3 complex is primarily driven by each protein’s transmembrane (TM) region, enforced through the interaction of evolutionarily conserved, charged, residues in each TM region (4, 5, 12). What, if any, role interactions between TCR and CD3 ECDs play in the assembly and function of the complex remains controversial (5): there are several plausible proposed models of activation, which are not necessarily mutually exclusive (5, 17–19). Although structures of TCR–peptide–MHC (pMHC) complexes (2), TCR–MHC-I–like complexes (1), and the CD3 dimers (6–10) have been separately determined, how the αβ TCR associates with the CD3 complex is largely unknown. Here, we use two independent structural approaches to gain an understanding of the TCR–CD3 complex organization and structure.     

CD14-expressing cancer cells establish the inflammatory and proliferative tumor microenvironment in bladder cancer

Nonresolving chronic inflammation at the neoplastic site is consistently associated with promoting tumor progression and poor patient outcomes. However, many aspects behind the mechanisms that establish this tumor-promoting inflammatory microenvironment remain undefined. Using bladder cancer (BC) as a model, we found that CD14-high cancer cells express higher levels of numerous inflammation mediators and form larger tumors compared with CD14-low cells. CD14 antigen is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein and has been shown to be critically important in the signaling pathways of Toll-like receptor (TLR). CD14 expression in this BC subpopulation of cancer cells is required for increased cytokine production and increased tumor growth. Furthermore, tumors formed by CD14-high cells are more highly vascularized with higher myeloid cell infiltration. Inflammatory factors produced by CD14-high BC cells recruit and polarize monocytes and macrophages to acquire immune-suppressive characteristics. In contrast, CD14-low BC cells have a higher baseline cell division rate than CD14-high cells. Importantly, CD14-high cells produce factors that further increase the proliferation of CD14-low cells. Collectively, we demonstrate that CD14-high BC cells may orchestrate tumor-promoting inflammation and drive tumor cell proliferation to promote tumor growth.Solid tumors represent a complex mass of tissue composed of multiple distinct cell types (1, 2). Cells within the tumor produce a range of soluble factors to create a complex of signaling networks within the tumor microenvironment (3–7). One of the outcomes of this crosstalk is tumor-promoting inflammation (TPI) (8, 9). TPI can modulate the functions of tumor-infiltrating myeloid lineage cells including macrophages (10–12). Tumor-associated macrophages (TAMs) consistently display an alternatively activated phenotype (M2) commonly found in sites of wound healing (13–18). These macrophages promote tumor growth while suppressing the host immune response locally (19–22). Polarization and subversion of tumor-infiltrating macrophages is accomplished via immune mediators in the tumor microenvironment (23, 24). Adding to the complexity of solid tumors is the heterogeneity of the cancer cells (2). Tumor cells of varying differentiation states and different characteristics coexist within a tumor (25–29). However, the different roles of each tumor cell subset during cancer progression remain undefined.Bladder cancer (BC) represents a growing number of solid tumors characterized by the infiltration of a significant number of myeloid cells in the neoplastic lesion (30, 31). We have previously determined that keratin 14 (KRT14) expression marks the most primitive differentiation state in BC cells (32). KRT14 expression is significantly associated with poor overall patient survival. However, the mechanisms used by KRT14-expressing cells to promote tumor growth remain unclear. In the current study, we found that KRT14+ basal BC cells also express higher levels of CD14. Here, we investigate the strategies used by KRT14+ CD14-high BC cells to promote tumor growth.