T-cell Receptor Complex Research Articles

Multiple mechanisms for TRAF3-mediated regulation of the T cell costimulatory receptor GITR

Tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) plays context-specific roles in multiple receptor-mediated signaling pathways in different cell types. Mice lacking TRAF3 in T cells display defective T-cell-mediated immune responses to immunization and infection and demonstrate defective early signaling via the TCR complex. However, the role of TRAF3 in the function of GITR/TNFRSF18, an important costimulatory member of the TNFR superfamily, is unclear. Here we investigated the impact of T cell TRAF3 status on both GITR expression and activation of specific kinases in the GITR signaling pathway in T cells. Our results indicate that TRAF3 negatively regulates GITR functions in several ways. First, expression of GITR protein was elevated in TRAF3-deficient T cells, resulting from both transcriptional and posttranslational regulation that led to greater GITR transcript levels, as well as enhanced GITR protein stability. TRAF3 associated with T cell GITR in a manner dependent upon GITR ligation. TRAF3 also inhibited several events of the GITR mediated early signaling cascade, in a manner independent of recruitment of phosphatases, a mechanism by which TRAF3 inhibits signaling through several other cytokine receptors. These results add new information to our understanding of GITR signaling and function in T cells, which is relevant to the potential use of GITR to enhance immune therapies.

Class I PI3K Provide Lipid Substrate in T Cell Autophagy Through Linked Activity of Inositol Phosphatases.

Autophagy, a highly conserved intracellular process, has been identified as a novel mechanism regulating T lymphocyte homeostasis. Herein, we demonstrate that both starvation- and T cell receptor-mediated autophagy induction requires class I phosphatidylinositol-3 kinases to produce PI(3)P. In contrast, common gamma chain cytokines are suppressors of autophagy despite their ability to activate the PI3K pathway. T cells lacking the PI3KI regulatory subunits, p85 and p55, were almost completely unable to activate TCR-mediated autophagy and had concurrent defects in PI(3)P production. Additionally, T lymphocytes upregulate polyinositol phosphatases in response to autophagic stimuli, and the activity of the inositol phosphatases Inpp4 and SHIP are required for TCR-mediated autophagy induction. Addition of exogenous PI(3,4)P2 can supplement cellular PI(3)P and accelerate the outcome of activation-induced autophagy. TCR-mediated autophagy also requires internalization of the TCR complex, suggesting that this kinase/phosphatase activity is localized in internalized vesicles. Finally, HIV-induced bystander CD4+ T cell autophagy is dependent upon PI3KI. Overall, our data elucidate an important pathway linking TCR activation to autophagy, via induction of PI3KI activity and inositol phosphatase upregulation to produce PI(3)P.

Application of Genome-Editing Technologies for Off-the-Shelf T Cell-Based Cancer Immunotherapy

: Genetically, engineered T-cell therapy is a personalized treatment that has demonstrated considerable therapeutic potential for solid tumors and hematopoietic cancers. However, endogenous T-cell receptors (TCRs) on the surface of engineered T cells hamper their application in the allogeneic settings by inducing graft-versus-host disease, where endogenous TCRs on the surface of engineered T cells respond to the recipient’s tissues. Since the cause of this allogeneic response is the TCR complex on the surface of the engineered T cells, preventing the expression of TCR components on the surface of these cells is a promising strategy to address this challenge. This review discusses the production of allogeneic chimeric antigen receptor T cells using genome-editing methods.

Abstract 1646: Development of small-molecule HPK1 inhibitors to unleash tumor-specific T cell responses

Abstract Hematopoietic progenitor kinase 1 (HPK1) is an intracellular protein kinase that negatively regulates T cell signaling and proliferation. Upon T cell receptor (TCR) activation, active HPK1 phosphorylates the adaptor protein SLP76 in the TCR complex, recruiting the negative regulator 14-3-3 and targeting components of the TCR signaling complex for degradation. HPK1 thus limits the TCR signaling important for mounting an effective immune response against tumor cells. We are employing structure-guided drug design to develop potent small-molecule inhibitors of HPK1. Our compounds potently inhibit HPK1 in biochemical assays, reduce levels of phosphorylated SLP76 and concomitantly increase IL-2 production by Jurkat T cells. Importantly, our HPK1 inhibitors enhance cytokine production by human and mouse primary T cells above that observed with TCR activation alone. Treatment of mice with orally available HPK1 inhibitors results in increased activation of antigen-specific CD8+ T cells in vivo and decreased tumor growth as single agent and in combination with clinically relevant checkpoint inhibitor antibodies. Our work confirms the importance of HPK1 for T cell function and supports HPK1 as a promising next generation immuno-oncology target. Citation Format: George Katibah, Yamini Ohol, Cyril Bucher, Lavanya Adusumilli, Deepika Kaveri, Omar Robles, Michael Sun, Cynthia Cho, Heather Milestone, Rachel Ames, Scott Jacobson, Dan Nebalasca, Justy Gomez- Guagua, Jerick Sanchez, Molly Grandcolas, Steve Wong, Martin Brovarney, Chandru Ramana, Thant Zaw, Lan Nguyen, Parcharee Tivitmahaisoon, Andrew Ng, Anqi Ma, Blanca Gomez, Michelle Ko, Paul Leger, Jeffrey Jackson, Grant Shibuya, Anton Shakhmin, Delia Bradford, Mengshu Xu, Mikhail Zibinsky, Daniel Poon, David Wustrow, Paul Kassner, Dirk Brockstedt. Development of small-molecule HPK1 inhibitors to unleash tumor-specific T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1646.

Abstract 1514: Engineering off-the-shelf T Cell Receptor Fusion Construct (TRuC) T cells

Abstract The natural T cell receptor (TCR) recognizes its cognate peptide antigen only when presented on human leukocyte antigen (HLA) molecules and requires HLA matching of α/β TCR-engineered T cells for cancer therapy. To bypass the need for HLA matching, we have previously described the generation of autologous T Cell Receptor Fusion Construct (TRuC™) T cells which are engineered to express a fusion protein that comprises an antibody-derived binder tethered to the CD3ϵ signaling subunit. Upon integration of the TRuC into the TCR, it recognizes tumor surface antigens independent of HLA and uses the complete receptor complex to trigger a comprehensive T cell response. Here, we report the engineering of off-the-shelf TRuC-T cells directed against CD19 and mesothelin, respectively. To eliminate the alloreactivity of α/β T cells and reduce the risk of graft-versus-host-disease (GvHD), the TRAC locus is knocked-out. To enable the re-assembly of the TCR, the endogenous TCRα and β subunits are replaced with fusion proteins comprising antibody binders fused to the murine TCRα or β constant domains, or alternatively, to the TCRγ and δ constant domains. Similar to their autologous counterparts, allogeneic CD19 and MSLN-targeting TRuC-T cells upregulate activation markers, secrete cytokines, and lyse tumor cells in an antigen-specific manner. Importantly, TRuC-T cells lack alloreactivity as demonstrated in mixed lymphocyte reactions and clear tumors in NSG xenograft model comparable to autologous anti-CD19 TRuC-T cells, but without signs of GvHD. Likewise, the functional activity and efficacy of allogeneic TRuC-T cells directed against mesothelin is similar to that of autologous anti-MSLN TRuC-T cells. To reduce host rejection and enhance the persistence of the allogeneic TRuC-T cells, MHC class I expression on the surface of the TRuC-T cells was down-regulated by means of RNAi. In summary we have engineered allogeneic TRuC-T cells that maintain the signaling properties of the TCR complex with comparable efficacy as donor-matched autologous TRuC-T cells; moreover, these T cells have the potential to persist in an allogeneic host by diminishing the risk of rejection by the host. Citation Format: Julie Donaghey, Philippe Kieffer-Kwon, Julio Gomez-Rodriguez, Troy Patterson, Jessica Gierut, Tiffany Chan, Ryan Milione, Joshua Daniel, Holly Horton, Robert Tighe, Robert Hofmeister, Dario A. Gutierrez. Engineering off-the-shelf T Cell Receptor Fusion Construct (TRuC) T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1514.

Allosteric activation of Tcell antigen receptor signaling by quaternary structure relaxation.

SummaryThe mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation.

TRAF3 in T Cells Restrains Negative Regulators of LAT to Promote TCR/CD28 Signaling.

The adaptor protein TNFR-associated factor 3 (TRAF3) is required for in vivo T cell effector functions and for normal TCR/CD28 signaling. TRAF3-mediated enhancement of TCR function requires engagement of both CD3 and CD28, but the molecular mechanisms underlying how TRAF3 interacts with and impacts TCR/CD28-mediated complexes to enhance their signaling remains an important knowledge gap. We investigated how TRAF3 is recruited to, and regulates, CD28 as a TCR costimulator. Direct association with known signaling motifs in CD28 was dispensable for TRAF3 recruitment; rather, TRAF3 associated with the CD28-interacting protein linker of activated T cells (LAT) in human and mouse T cells. TRAF3-LAT association required the TRAF3 TRAF-C domain and a newly identified TRAF2/3 binding motif in LAT. TRAF3 inhibited function of the LAT-associated negative regulatory protein Dok1, which is phosphorylated at an inhibitory tyrosine residue by the tyrosine kinase breast tumor kinase (Brk/PTK6). TRAF3 regulated Brk activation in T cells, limiting the association of protein tyrosine phosphatase 1B (PTP1B) with the LAT complex. In TRAF3-deficient cells, LAT complex-associated PTP1B was associated with dephosphorylation of Brk at an activating tyrosine residue, potentially reducing its ability to inhibit Dok1. Consistent with these findings, inhibiting PTP1B activity in TRAF3-deficient T cells rescued basal and TCR/CD28-mediated activation of Src family kinases. These results reveal a new mechanism for promotion of TCR/CD28-mediated signaling through restraint of negative regulation of LAT by TRAF3, enhancing the understanding of regulation of the TCR complex.

Abstract 1528: Discovery and preclinical characterization of fratricide-resistant TRuC T-cells targeting CD70

Abstract Previously we have described the design and antitumor activity of T cell receptor fusion constructs (TRuC™) that tether an antibody-derived binder to one of the TCR subunits to achieve redirected T cell killing of tumor cells independent of HLA. Different from CAR-T cells, TRuCs are integrated into the full TCR complex and thus harness its full signaling capacity. The cell surface antigen CD70 represents a promising target for cancer immunotherapy for its selective overexpression in various hematological and solid tumor indications. Because the normal tissue expression of CD70 occurs on activated lymphocytes, including activated T cells, fratricide (self-killing) has been recognized as a significant challenge for CD70-targeted T cell therapies. To address this challenge, we discovered a diverse pool of fully human anti-CD70 scFv binders that were used to make TRuC-T cells and then functionally screened for fratricide-resistance in vitro. We successfully identified a CD70-targeted TRuC-T cell candidate that exhibits normal T cell expansion and an improved memory phenotype, clearly differentiating from fratricide-prone candidates, all while maintaining potent cytotoxicity and cytokine production against tumor cells expressing both low and high levels of CD70. In addition, our CD70-targeted TRuC-T cells showed significant anti-tumor efficacy in multiple xenograft mouse models with no evidence of in vivo fratricide. In summary, we have engineered a fratricide-resistant CD70-directed TRuC-T cell therapy that has the potential to treat a wide range of both hematologic and solid cancers. Citation Format: Jian Ding, Amy Watt, Erica Liu, Zieba Adam, Derrick McCarthy, Jessica Gierut, Brett Schrand, Michael Lofgren, Jason Lajoie, Vania Kenanova, Philippe Kiefer-Kwon, Holly Horton, Dario A. Gutierrez, Robert Hofmeister, Robert Tighe. Discovery and preclinical characterization of fratricide-resistant TRuC T-cells targeting CD70 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1528.

Abstract CT105: Preliminary safety and efficacy of gavocabtagene autoleucel (gavo-cel, TC-210), a T cell receptor fusion construct (TRuC™), in patients with treatment refractory mesothelin overexpressing solid tumors

Abstract Background: Mesothelin overexpression is present in over 80,000 patients/year with either malignant mesothelioma (MPM), ovarian cancer, cholangiocarcinoma, or non-small cell lung cancer (NSCLC) in the U.S. alone. We are testing a novel genetically engineered anti-mesothelin TRuC™ T cell therapy called gavo-cel (TC-210) in a Phase 1 study in treatment refractory patients with any of the four aforementioned cancers (NCT03907852). Methods: Gavo-cel was engineered by transducing T cells with a lentiviral vector encoding for an anti-mesothelin llama-derived single domain antibody fused to the CD3epsilon subunit using a flexible glycine serine sequence. Upon translation, gavo-cel TRuCs integrate and reprogram intact TCR complexes to recognize tumor surface mesothelin in an HLA-independent manner. Eligibility criteria required demonstration of mesothelin overexpression on tumors (≥50% of tumor cells expressing 2+ or 3+ staining by IHC) at a central laboratory. Gavo-cel dose escalation proceeded following a modified 3+3 schema. TC-210 expansion and serum cytokine levels were serially measured. Results: Seven patients (6 MPM, 1 ovarian) received a single gavo-cel intravenous infusion at the initial dose of 5x107/m2 either alone (dose level [DL] 0, n=1) or following lymphodepletion (LD) (DL1, n=6) with fludarabine (30mg/m2/day x4) and cyclophosphamide (600mg/m2/day x3). The median age was 69 years (range, 36-84) and the median number of prior therapies was 5 (range, 2-9). Four patients had received ≥4 lines of therapy, including immune checkpoint inhibitors (n=5), the anti-mesothelin ADC anetumab ravtansine (n=1), and anti-mesothelin mRNA CAR-T (n=1). Four patients required bridging therapy. No on-target/off-tumor toxicities or neurotoxicity events of any grade were observed. Grade (gr) ≥3 treatment emergent AEs occurred in 2 (29%) patients with MPM treated at DL1: 1 with gr3 CRS, and 1 with gr3 CRS, gr3 pneumonitis and gr5 fungal sepsis (unrelated to gavo-cel). CRS and pneumonitis events resolved with tocilizumab and corticosteroids. Grade 3 pneumonitis was considered a DLT, resulting in DL1 cohort expansion to 6 patients. At data cut-off date (November 8, 2020), 6 patients have been followed for >3 months, including 4 followed through month 6. The disease control rate was 100%. All patients experienced target lesion regression by CT: median 61% (range, 5-75%). Three (43%) patients (2 with mesothelioma and 1 with ovarian cancer) had a partial response by RECIST v1.1 criteria, including 1 achieving a complete metabolic response. After a median follow-up of 6.5 months, the median OS at 6 months was (86%). The addition of LD increased TC-210 peak expansion and persistence, with cells being detected up to 30 days post infusion in peripheral blood. Conclusion: Intravenous systemic administration of gavo-cel was generally safe and resulted in three of seven patients having objective partial response. Dose escalation is ongoing at 1x108/m2. Updated clinical and translational data will be presented. Citation Format: David S. Hong, Melissa Johnson, Janos L. Tanyi, Lauren MacMullen, Robert Tighe, Lizzy Jalbert, Viera P. Muzithras, Kevin Zikaras, Alfonso Quintas Cardama, Raffit Hassan. Preliminary safety and efficacy of gavocabtagene autoleucel (gavo-cel, TC-210), a T cell receptor fusion construct (TRuC™), in patients with treatment refractory mesothelin overexpressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT105.

Immune Gene Expression Profiling in Children and Adults with Acute Myeloid Leukemia Identifies Distinct Phenotypic Patterns

Therapeutic strategies in patients with acute myeloid leukemia (AML) have remained relatively unchanged over the last 30 years. AML is only cured in 35-40% of patients <60 years of age and in 5-15% of patients >60 years of age. Elderly patients who are unfit to receive intensive salvage regimens can only be offered low-intensity therapy or best supportive care. In children, AML accounts for 20% of leukemias. Although intensive multi-agent chemotherapy in conjunction with improved supportive care has increased survival rates to 60-70%, 30-40% of children with AML relapse and only one-third of them will survive to adulthood. The development and delivery of new therapeutic strategies for high-risk AML, including immunotherapy, therefore remains a priority.Tumor phenotypes are dictated not only by the neoplastic cell component, but also by the tumor microenvironment (TME), which includes immune and inflammatory cells and mediators. We have previously shown that the treatment of primary AML cells with interferon (IFN)-γ in vitro induces functional indoleamine 2,3-dioxygenase-1 (IDO1), a molecule with potent immunosuppressive properties, in 50% of unselected cases (‘IFN-γ responders‘). With a median follow-up of 8 years, these IFN-γ responders experienced a significantly shorter event-free survival and overall survival.This multi-institutional study was undertaken to collect comprehensive immune profiles across the biological heterogeneity of childhood and adult AML, with the aim to implement new molecularly targeted agents for patients with specific immunologic subtypes of AML. We employed the nCounter™ system (NanoString Technologies, Seattle, USA), a medium-throughput, fully automated digital platform, to characterize bone marrow (BM) specimens from 70 patients with non-promyelocytic AML (42 children and 28 adults). Ninety BM samples (63 from patients with de novo AML, 18 from patients with AML in complete remission [CR] and 9 from patients with relapsed AML) were analyzed on the nCounter® FLEX platform, using the RNA Pan-Cancer Immune Profiling Panel. This panel detects the expression of genes for 109 cell surface markers on 24 immune cell types, 30 cancer testis antigens, and >500 immune response genes. Transcriptomic data were normalized using the nSolver™ software package (alpha version 4.0). Patient age and status (disease onset, achievement of CR and disease relapse) were selected as covariates.Hierarchical clustering identified patient subgroups with heightened expression of CD8 T-cell, T helper type 1 (Th1)-cell, B-cell and cytotoxicity-related genes, including genes related to natural killer (NK)-cell function, in the leukemia microenvironment (“immune enriched” patients) (Fig. 1A). Interestingly, a select group of patients over-expressed genes associated with monocyte/macrophage, neutrophil and mast cell functions, with low expression levels of T-cell and B-cell genes. AML patients with ‘immune enriched’ gene profiles also expressed CD8A , IFNG , FOXP3 , the T-cell chemoattractants CXCL9 , CXCL10, and inhibitory molecules including IDO1 and the immune checkpoints LAG3 , CTLA4 and PDL1 . This immune landscape is consistent with a ‘T-cell inflamed’ phenotype that might underpin the establishment of adaptive immune resistance and immune escape. Gene set analyses identified differences in immune gene expression levels between childhood and adult AMLs, as well as patients with newly diagnosed AML, relapsed AML or AML in CR (Fig. 1B-C). In particular, genes highly expressed in adult AMLs compared with childhood disease included chemokine, dendritic cell and macrophage genes . In contrast, immune genes down-regulated in adult compared with childhood AMLs included integrin family members, cytokine receptors, T-cell receptor complex components (CD3EAP ) and lipocalin-2 (LCN2 ), a neutrophil gelatinase-associated molecule implicated in suppression of tumor invasiveness and metastasis.In conclusion, our analysis has identified heterogeneous immunological profiles in children and adults with AML at different disease stages. From a clinical standpoint, ‘immune enriched’ AMLs might be amenable to immunotherapy approaches tailored to the BM microenvironment, including blockade of co-inhibitory molecules and/or small-molecule IDO1 inhibitors.Grant support: Roger Counter Foundation, UK; Qatar National Research Fund (#NPRP8-2297-3-494). [Display omitted] DisclosuresWarren:NanoString Technologies: Employment, Equity Ownership. Hood:NanoString Technologies: Employment, Equity Ownership. Danaher:NanoString Technologies: Employment, Equity Ownership. Cesano:NanoString Technologies: Employment, Equity Ownership. Beechem:NanoString Technologies: Employment, Equity Ownership. Tasian:Gilead Sciences, Inc.: Research Funding; Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rutella:NanoString Technologies, Seattle, USA: Research Funding.

Cooperative ectodomain interaction among TCRαβ, CD3δɛ and CD3γɛ

Abstract The T-cell receptor (TCR) complex comprises of the ligand-binding subunit TCRαβ, and the signaling subunits CD3δɛ, CD3γɛ and CD3ζζ, with the Cα/Cβ in proximity to both CD3δɛ and CD3γɛ extracellularly. Direct measurements of the ectodomain interactions had not been successful, although they are believed to be important for TCR stability and functionality. Mechanical force has been shown to modulate TCR–ligand interactions. The TCR mechanosensor hypothesis predicts that force-encoded information may transmit from pMHC to CD3 via TCR-CD3 interaction. Evaluating ectodomain interactions among TCRαβ, CD3δɛ and CD3γɛ can help elucidate the TCR triggering mechanism and further guide the design of TCR-based immunotherapy. Using two mechanical based assays, we were able to measure the weak two-dimensional (2D) affinities among ectodomains of human TCRαβ (2B4-LC13), and human CD3δɛ or CD3γɛ and showed catch bond formation where lifetimes of TCRαβ–CD3δɛ and TCRαβ–CD3γɛ bonds are prolonged by forces &amp;lt;15 pN. Remarkably, CD3δɛ and CD3γɛ bind TCRαβ cooperatively, forming more bonds that last longer when both CD3s interact with TCRαβ as a whole than the sum of either CD3 interacting with TCRαβ individually. Interestingly, these measurements are comparable to 2D affinity and force-dependent bond lifetime of the 2B4 TCR interaction with its cognate ligand K5:I-Ek, supporting their relevance to TCR function. Using molecular dynamics simulations based on a published Cryo-EM structure, we identified the formation of long-lasting CD3δɛ–TCRαβ–CD3γɛ trimolecular bonds as the structural mechanisms of the cooperativity. Our work helps explain TCR function under force and suggests strategies for engineering of TCR for immunotherapy applications.

Decreased Levels of STAT1 and Interferon-γ-Induced STAT1 Phosphorylation in Rheumatoid Arthritis CD4 and CD8 T Cells.

ObjectiveWe investigated whether a previously reported association of IFNGR expression with rheumatoid arthritis (RA) and its radiographic severity reflects differences in proximal interferon‐γ (IFN‐γ) signaling in T cells from patients with RA compared with healthy controls (HC).MethodsUsing phosphoflow cytometry, we compared IFN‐γ–stimulated signal transducer and activator of transcription 1 (STAT1) activation in CD4+ and CD8+ T‐cell populations from patients with RA and HC.ResultsCompared with controls, patients with RA had a higher proportion of CD4+ T cells, associated with expansion of the CD4+ effector memory subset. Several CD4+ T‐cell types exhibited reduced IFN‐γ–induced phosphoSTAT1Y701 (pSTAT1Y701) in patients with RA compared with HC. Engaging the T‐cell receptor (TCR) complex on CD4+ T cells during IFN‐γ stimulation abrogated the reduction in STAT1 activation in patients with RA but had no effect in HC. The phosphorylation of STAT1S727 was similar in CD4+ T cells from patients with RA and HC. In contrast to CD4+ T cells, IFN‐γ–induced pSTAT1Y701 levels in CD8+ T cells were equivalent or higher in patients with RA compared with HC. Total STAT1 levels (phosphorylated + unphosphorylated) were lower in CD4+ and CD8+ T cells from patients with RA compared with HC.ConclusionWe report diminished IFN‐γ–induced pSTAT1Y701 levels in CD4+ T cells in patients with RA, which were restored by TCR engagement. There were lower levels of total STAT1 in patients with RA compared with HC, but this likely does not explain diminished IFN‐γ–induced pSTAT1Y701 levels in CD4+ T cells because activation in CD8+ T cells was higher or equivalent to that seen in HC. The enhanced IFNGR expression in patients with RA reported previously may reflect a compensatory mechanism to overcome deficiency in IFN‐γ responsiveness.

How the Discovery of the CD4/CD8-p56lck Complexes Changed Immunology and Immunotherapy.

The past 25 years have seen enormous progress in uncovering the receptors and signaling mechanisms on T-cells that activate their various effecter functions. Until the late 1980s, most studies on T-cells had focused on the influx of calcium and the levels of cAMP/GMP in T-cells. My laboratory then uncovered the interaction of CD4 and CD8 co-receptors with the protein-tyrosine kinase p56lck which are now widely accepted as the initiators of the tyrosine phosphorylation cascade leading to T-cell activation. The finding explained how immune recognition receptors expressed by many immune cells, which lack intrinsic catalytic activity, can transduce activation signals via non-covalent association with non-receptor tyrosine kinases. The discovery also established the concept that a protein tyrosine phosphorylation cascade operated in T-cells. In this vein, we and others then showed that the CD4- and CD8-p56lck complexes phosphorylate the TCR complexes which led to the identification of other protein-tyrosine kinases such as ZAP-70 and an array of substrates that are now central to studies in T-cell immunity. Other receptors such as B-cell receptor, Fc receptors and others were also subsequently found to use src kinases to control cell growth. In T-cells, p56lck driven phosphorylation targets include co-receptors such as CD28 and CTLA-4 and immune cell-specific adaptor proteins such as LAT and SLP-76 which act to integrate signals proximal to surface receptors. CD4/CD8-p56lck regulated events in T-cells include intracellular calcium mobilization, integrin activation and the induction of transcription factors for gene expression. Lastly, the identification of the targets of p56lck in the TCR and CD28 provided the framework for the development of chimeric antigen receptor (CAR) therapy in the treatment of cancer. In this review, I outline a history of the development of events that led to the development of the “TCR signaling paradigm” and its implications to immunology and immunotherapy.

Potentiating CD8+ T cell antitumor activity by inhibiting PCSK9 to promote LDLR-mediated TCR recycling and signaling

Metabolic regulation has been proven to play a critical role in T cell antitumor immunity. However, cholesterol metabolism as a key component of this regulation remains largely unexplored. Herein, we found that the low-density lipoprotein receptor (LDLR), which has been previously identified as a transporter for cholesterol, plays a pivotal role in regulating CD8+ T cell antitumor activity. Besides the involvement of cholesterol uptake which is mediated by LDLR in T cell priming and clonal expansion, we also found a non-canonical function of LDLR in CD8+ T cells: LDLR interacts with the T-cell receptor (TCR) complex and regulates TCR recycling and signaling, thus facilitating the effector function of cytotoxic T-lymphocytes (CTLs). Furthermore, we found that the tumor microenvironment (TME) downregulates CD8+ T cell LDLR level and TCR signaling via tumor cell-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) which binds to LDLR and prevents the recycling of LDLR and TCR to the plasma membrane thus inhibits the effector function of CTLs. Moreover, genetic deletion or pharmacological inhibition of PCSK9 in tumor cells can enhance the antitumor activity of CD8+ T cells by alleviating the suppressive effect on CD8+ T cells and consequently inhibit tumor progression. While previously established as a hypercholesterolemia target, this study highlights PCSK9/LDLR as a potential target for cancer immunotherapy as well.

Differential Analysis of T-Cell Activation by a Library of Membrane-Tethered, Monovalent, Fab’-Based Synthetic T-Cell Receptor Agonists

T cells mediate adaptive immunity by identifying pathogenic peptide-major histocompatibility complex (pMHC) molecules presented on the surface of antigen presenting cells (APCs). The T cell-APC junction, where T-cell receptors (TCRs) bind pMHC, is necessary for productive TCR triggering; T cells activate in response to single-molecule densities of membrane-bound agonist pMHC, but they do not respond to pMHC in solution. Crosslinked pMHC or multivalent antibodies can be used to activate T cells from solution, but these widely used strategies produce different activation profiles than natural, monovalent, membrane-associated pMHC. Here, we introduce a library of strictly monovalent Fab’ fragments that bind three distinct epitopes on the TCR complex and activate T cells when coupled to a supported lipid bilayer via DNA complementation. Like pMHC, these synthetic Fab’-based ligands are inactive from solution. I will discuss the differential activation profiles of T cells in response to the library of ligands and the insights these data provide about the physiochemical and mechanical requirements for productive TCR triggering. The intrinsically modular nature of the hybrid Fab’-DNA ligands facilitates control of numerous physical aspects of the TCR triggering process to explore T-cell activation by synthetic ligands. Notably, the Fab's used can activate polyclonal populations of T cells, and so they can be used in studies for which T-cell activation by pMHC is not possible. These studies additionally have potential to inform the design of cancer immunotherapies such as bispecific T cell engagers (BiTEs), which are structurally similar to the synthetic Fab’ ligands.

Mechanical Response of the A6 TCR in Complex with PMHC with and without Load

The αβ T-Cell Receptor complex (αβTCR) contains a ligand-binding heterodimer (TCRαβ) along with signaling CD3 co-receptors. Previous studies have shown that upon binding to a peptide-bound major histocompatibility complex (pMHC), TCRαβ acts as a mechanosensor via a “catch bond” behavior where the TCRαβ-pMHC bond lifetime increases under piconewton level force. We have recently found that load suppresses relative motion between Vα and Vβ variable domains that contain pMHC-binding complementarity determining region (CDR) loops.

A Multimodal Platform for Simultaneous T-Cell Imaging, Defined Activation, and Mechanobiological Characterization.

T-cell antigen recognition is accompanied by extensive morphological rearrangements of the contact zone between the T-cell and the antigen-presenting cell (APC). This process involves binding of the T-cell receptor (TCR) complex to antigenic peptides presented via MHC on the APC surface, the interaction of costimulatory and adhesion proteins, remodeling of the actin cytoskeleton, and the initiation of downstream signaling processes such as the release of intracellular calcium. However, multiparametric time-resolved analysis of these processes is hampered by the difficulty in recording the different readout modalities at high quality in parallel. In this study, we present a platform for simultaneous quantification of TCR distribution via total internal reflection fluorescence microscopy, of intracellular calcium levels, and of T-cell-exerted forces via atomic force microscopy (AFM). In our method, AFM cantilevers were used to bring single T-cells into contact with the activating surface. We designed the platform specifically to enable the study of T-cell triggering via functionalized fluid-supported lipid bilayers, which represent a widely accepted model system to stimulate T-cells in an antigen-specific manner. In this paper, we showcase the possibilities of this platform using primary transgenic T-cells triggered specifically via their cognate antigen presented by MHCII.

Study of Signal Transduction Pathways by Phospho-Protein Evaluation.

Flow cytometric evaluation of phosphorylation status of signal transduction molecules is a useful method to study T-cell signaling pathways. As mutations occurring in TCR complex molecules, common gamma chain family's cytokines, their receptors or molecules involved in these pathways can lead to severe immune system defects, the study of T-cell signal transduction can be applied to both basic and clinical/translational research areas. In the present chapter, we show two different protocols for the study of T- cell response to an antigen-like stimulus and to IL-2.

Monoclonal Antibodies, Bispecific Antibodies and Antibody-Drug Conjugates in Oncohematology.

The therapeutic outcomes and the prognosis of patients with various hematologic malignancies are not always ideal with the current standard of care. The aim of this study is to analyze the results of the use of monoclonal antibodies, bispecific antibodies and antibody-drug conjugates for the therapy of malignant hemopathies. A mini-review was achieved using the articles published in Web of Science and PubMed between January 2017 and January 2020 and the new patents were made in this field. Naked monoclonal antibodies have improved the therapeutic results obtained with standard of care, but they also have side effects and the use of some of them can lead to the loss of the target antigen through trogocytosis, which explains the resistance that occurs during therapy. The results obtained with naked monoclonal antibodies have been improved by a better monoclonal antibody preparation, the use of bispecific antibodies (against two antigens on the target cell surface or by binding both surface antigen on target cells and T-cell receptor complex, followed by cytotoxic T-lymphocytes activation and subsequent cytolysis of the target cell), the use of monoclonal or bispecific constructs in frontline regimens, combining immunotherapy with chemotherapy, including through the use of antibody-drug conjugates (which provides a targeted release of a chemotherapeutic agent). Immunotherapy and immuno-chemotherapy have improved the outcome of the patients with malignant hemopathies through a targeted, personalized therapy, with reduced systemic toxicity, which in some cases can even induce deep complete remissions, including minimal residual disease negativity.

A single-domain bispecific antibody targeting CD1d and the NKT T-cell receptor induces a potent antitumor response.

Antibody-mediated modulation of major histocompatibility complex (MHC) molecules, or MHC class I-like molecules, could constitute an effective immunotherapeutic approach. We describe how single-domain antibodies (VHH), specific for the human MHC class I-like molecule CD1d, can modulate the function of CD1d-restricted T cells and how one VHH (1D12) specifically induced strong type I natural killer T (NKT) cell activation. The crystal structure of the VHH1D12-CD1d(α-GalCer)-NKT T-cell receptor (TCR) complex revealed that VHH1D12 simultaneously contacted CD1d and the type I NKT TCR, thereby stabilizing this interaction through intrinsic bispecificity. This led to greatly enhanced type I NKT cell-mediated antitumor activity in in vitro, including multiple myeloma and acute myeloid leukemia patient-derived bone marrow samples, and in vivo models. Our findings underscore the versatility of VHH molecules in targeting composite epitopes, in this case consisting of a complexed monomorphic antigen-presenting molecule and an invariant TCR, and represent a generalizable antitumor approach.