T-cell Receptor Engagement Research Articles

Abstract PS17-07: Impact of chemotherapy (chemo) on t-cell maturation and clonal proliferation in early-stage and metastatic breast cancer

Abstract Background Adaptive immunity is initiated by T-cell receptor (TCR) engagement with cognate tumor antigen, leading to T-cell maturation (i.e. conversion from naïve to effector state), clonal proliferation, and potentially tumor elimination. Therefore, broad T-cell repertoire diversity could be an important determinant of anti-tumor immunity. In breast cancer, chemo may facilitate adaptive immunity, but conversely may also be lymphotoxic. We characterize the impacts of curative-intent chemo and palliative combination chemo/immune checkpoint inhibition (ICI) on lymphocyte quantity, maturation, and clonal proliferation. Methods Peripheral blood mononuclear cells (PBMCs) were collected at baseline and serially in stage I-III subjects receiving curative-intent chemo (dose-dense doxorubicin, cyclophosphamide, paclitaxel, n=20), and in stage IV subjects receiving palliative ICI (pembrolizumab) plus chemo (paclitaxel, n=15; or capecitabine, n=14). Flow cytometry was conducted on fresh PBMCs to minimize cellular losses related to cryopreservation. DNA was extracted for T-cell clonality analysis using the immunoSEQ Assay (Adaptive Biotechnologies) at deep resolution. T-cell richness, calculated using the iChao1 richness estimator was used to assess overall T-cell diversity. Results Both dose-dense chemo and palliative chemo/ICI were lymphodepleting, however dose-dense chemo was associated with greater reductions in CD4+ naïve T cell count (week 8 counts 34% vs. 96% of baseline, p<.001) and T-cell richness (week 8 richness 57% vs 90% of baseline, p<.001). These effects were durable, with reductions in lymphocyte subsets (ALC, CD4, CD8, naïve, effector, and central memory subsets) and T-cell richness (estimated rearrangements 4.37 vs 3.29 x 105/sample) sustained at metastatic relapse. T-cell richness correlated with CD4+ naïve cell proportion (R2=0.52), suggesting a major contribution of CD4+ naïve cells to overall T-cell diversity. In this dataset, acute memory T-cell expansions were observed following initiation of chemo, but only among younger patients (age<60). Conclusions Curative-intent cytotoxic chemotherapy durably alters the quantity and diversity of peripheral T-cells, particularly of naïve cells, contributing to lymphopenia and reduced T-cell richness at metastatic relapse. Acute expansions of EM subsets were also observed but only in younger individuals. These contrasting immunologic effects highlight the potential importance of sequencing of ICI relative to chemo, and raise concerns regarding age-related thymic involution and immunosenescence. Ongoing research is warranted to investigate the predictive/prognostic utility of T-cell diversity/quantity in breast cancer, and whether thymic T-cell regeneration can be targeted therapeutically to enhance immunotherapy response, for example with cytokine therapies (IL-12 or IL-7, NCT04095689) or sex steroid inhibition (NCT03650894). Citation Format: Brie M Chun, Joanna Pucilowska, Valerie Conrad, Mark Schmidt, Isaac Kim, Paul Fields, Alison Conlin, Heather McArthur, David B Page. Impact of chemotherapy (chemo) on t-cell maturation and clonal proliferation in early-stage and metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-07.

Isolevuglandin-Modified Cardiac Proteins Drive CD4+ T-Cell Activation in the Heart and Promote Cardiac Dysfunction.

Despite the well-established association between T-cell-mediated inflammation and nonischemic heart failure, the specific mechanisms triggering T-cell activation during the progression of heart failure and the antigens involved are poorly understood. We hypothesized that myocardial oxidative stress induces the formation of isolevuglandin (IsoLG)-modified proteins that function as cardiac neoantigens to elicit CD4+ T-cell receptor (TCR) activation and promote heart failure. We used transverse aortic constriction in mice to trigger myocardial oxidative stress and T-cell infiltration. We profiled the TCR repertoire by mRNA sequencing of intramyocardial activated CD4+ T cells in Nur77GFP reporter mice, which transiently express GFP on TCR engagement. We assessed the role of antigen presentation and TCR specificity in the development of cardiac dysfunction using antigen presentation-deficient MhcII-/- mice and TCR transgenic OTII mice that lack specificity for endogenous antigens. We detected IsoLG protein adducts in failing human hearts. We also evaluated the role of reactive oxygen species and IsoLGs in eliciting T-cell immune responses in vivo by treating mice with the antioxidant TEMPOL and the IsoLG scavenger 2-hydroxybenzylamine during transverse aortic constriction, and ex vivo in mechanistic studies of CD4+ T-cell proliferation in response to IsoLG-modified cardiac proteins. We discovered that TCR antigen recognition increases in the left ventricle as cardiac dysfunction progresses and identified a limited repertoire of activated CD4+ T-cell clonotypes in the left ventricle. Antigen presentation of endogenous antigens was required to develop cardiac dysfunction because MhcII-/- mice reconstituted with CD4+ T cells and OTII mice immunized with their cognate antigen were protected from transverse aortic constriction-induced cardiac dysfunction despite the presence of left ventricle-infiltrated CD4+ T cells. Scavenging IsoLGs with 2-hydroxybenzylamine reduced TCR activation and prevented cardiac dysfunction. Mechanistically, cardiac pressure overload resulted in reactive oxygen species-dependent dendritic cell accumulation of IsoLG protein adducts, which induced robust CD4+ T-cell proliferation. Our study demonstrates an important role of reactive oxygen species-induced formation of IsoLG-modified cardiac neoantigens that lead to TCR-dependent CD4+ T-cell activation within the heart.

Differentiating MHC-Dependent and -Independent Mechanisms of Lymph Node Stromal Cell Regulation of Proinsulin-Specific CD8+ T Cells in Type 1 Diabetes.

Lymph node stromal cells (LNSC) are essential for providing and maintaining peripheral self-tolerance of potentially autoreactive cells. In type 1 diabetes, proinsulin-specific CD8+ T cells, escaping central and peripheral tolerance, contribute to β-cell destruction. Using G9Cα-/-CD8+ T cells specific for proinsulin, we studied the mechanisms by which LNSC regulate low-avidity autoreactive cells in the NOD mouse model of type 1 diabetes. Whereas MHC-matched NOD-LNSC significantly reduced G9Cα-/-CD8+ T-cell cytotoxicity and dendritic cell-induced proliferation, they failed to sufficiently regulate T cells stimulated by anti-CD3/CD28. In contrast, non-MHC-matched, control C57BL/6 mouse LNSC suppressed T-cell receptor engagement by anti-CD3/CD28 via MHC-independent mechanisms. This C57BL/6-LNSC suppression was maintained even after removal of the LNSC, demonstrating a direct effect of LNSC on T cells, modifying antigen sensitivity and effector function. Thus, our results suggest that a loss of NOD-LNSC MHC-independent suppressive mechanisms may contribute to diabetes development.

Peptide–MHC Binding Reveals Conserved Allosteric Sites in MHC Class I- and Class II-Restricted T Cell Receptors (TCRs)

T cells are vital for adaptive immune responses that protect against pathogens and cancers. The T cell receptor (TCR)–CD3 complex comprises a diverse αβ TCR heterodimer in noncovalent association with three invariant CD3 dimers. The TCR is responsible for recognizing antigenic peptides bound to MHC molecules (pMHC), while the CD3 dimers relay activation signals to the T cell. However, the mechanisms by which TCR engagement by pMHC is transmitted to CD3 remain mysterious, although there is growing evidence that mechanosensing and allostery both play a role. Here, we carried out NMR analysis of a human autoimmune TCR (MS2-3C8) that recognizes a self-peptide from myelin basic protein presented by the MHC class II molecule HLA-DR4. We observed pMHC-induced NMR signal perturbations in MS2-3C8 that indicate long-range effects on TCR β chain conformation and dynamics. Our results demonstrate that, in addition to expected changes in the NMR resonances of pMHC-contacting residues, perturbations extend to the Vβ/Vα, Vβ/Cβ, and Cβ/Cα interfacial regions. Moreover, the pattern of long-range perturbations is similar to that detected previously in the β chains of two MHC class I-restricted TCRs, thereby revealing a common allosteric pathway among three unrelated TCRs. Molecular dynamics (MD) simulations predict similar pMHC-induced effects. Taken together, our results demonstrate that pMHC binding induces long-range allosteric changes in the TCR β chain at conserved sites in both representative MHC class I- and class II-restricted TCRs, and that these sites may play a role in the transmission of signaling information.

The thioredoxin-1 inhibitor Txnip restrains effector T-cell and germinal center B-cell expansion.

Thioredoxin-1 (Trx1) is a vital component for cellular redox homeostasis. In Tcells, Trx1 donates electrons for the de novo synthesis of deoxyribonucleotides to allow rapid cell proliferation. The Trx-interacting protein (Txnip) binds to the reduced Trx1 and inhibits its activity. However, the role of Txnip in adaptive immunity in vivo is unknown. Here, we show that absence of Txnip increased proliferation of effector Tcells and GC B-cell responses in response to lymphocytic choriomeningitis virus and Qβ virus-like particles, respectively, but did not affect development and homeostasis of T and B cells. While downregulation of Txnip and concomitant upregulation of Trx1 is critical for rapid T-cell expansion upon viral infection, re-expression of Txnip and consequently inhibition of Trx1 is important to restrain late T-cell expansion. Importantly, we demonstrated that T-cell receptor (TCR) engagement but not CD28 costimulation is critically required for Txnip downregulation. Thus, this study further uncovers positive and negative control of lymphocyte proliferation by the Trx1 system.

Landscape of Exhausted Virus-Specific CD8 T Cells in Chronic LCMV Infection

A hallmark of chronic infections is the presence of exhausted CD8 Tcells, characterized by a distinct transcriptional program compared with functional effector or memory cells, co-expression of multiple inhibitory receptors, and impaired effector function, mainly driven by recurrent Tcell receptor engagement. In the context of chronic lymphocytic choriomeningitis virus (LCMV) infection in mice, most studies focused on studying splenic virus-specific CD8 Tcells. Here, we provide a detailed characterization of exhausted CD8 Tcells isolated from six different tissues during established LCMV infection, using single-cell RNA sequencing. Our data reveal that exhausted cells are heterogeneous, adopt organ-specific transcriptomic profiles, and can be divided into five main functional subpopulations: advanced exhaustion, effector-like, intermediate, proliferating, or memory-like. Adoptive transfer experiments showed that these phenotypes are plastic, suggesting that the tissue microenvironment has a major impact in shaping the phenotype and function of virus-specific CD8 Tcells during chronic infection.

CARD19, the protein formerly known as BinCARD, is a mitochondrial protein that does not regulate Bcl10-dependent NF-κB activation after TCR engagement

After T cell receptor (TCR) engagement, the CARD11-Bcl10-Malt1 (CBM) complex oligomerizes to transduce NF-κB activating signals. Bcl10 is then degraded to limit NF-κB activation. The cDNA AK057716 (BinCARD-1) was reported to encode a novel CARD protein that interacts with Bcl10 and modestly inhibits NF-κB activation. In a later study, a second isoform, BinCARD-2, was identified. Here, we report that the cDNA AK057716 (BinCARD-1) is an incompletely spliced derivative of the gene product of C9orf89, whereas CARD19 (BinCARD-2) represents the properly spliced isoform, with conservation across diverse species. Immunoblotting revealed expression of CARD19 in T cells, but no evidence of BinCARD-1 expression, and microscopy demonstrated that endogenous CARD19 localizes to mitochondria. Although we confirmed that both BinCARD-1 and CARD19 can inhibit NF-κB activation and promote Bcl10 degradation when transiently overexpressed in HEK293T cells, loss of endogenous CARD19 expression had little effect on Bcl10-dependent NF-κB activation, activation of Malt1 protease function, or Bcl10 degradation after TCR engagement in primary murine CD8 T cells. Together, these data indicate that the only detectable translated product of C9orf89 is the mitochondrial protein CARD19, which does not play a discernible role in TCR-dependent, Bcl10-mediated signal transduction to Malt1 or NF-κB.

LymphoAtlas: a dynamic and integrated phosphoproteomic resource of TCR signaling in primary T cells reveals ITSN 2 as a regulator of effector functions

T‐cell receptor (TCR) ligation‐mediated protein phosphorylation regulates the activation, cellular responses, and fates of T cells. Here, we used time‐resolved high‐resolution phosphoproteomics to identify, quantify, and characterize the phosphorylation dynamics of thousands of phosphorylation sites in primary T cells during the first 10 min after TCR stimulation. Bioinformatic analysis of the data revealed a coherent orchestration of biological processes underlying T‐cell activation. In particular, functional modules associated with cytoskeletal remodeling, transcription, translation, and metabolic processes were mobilized within seconds after TCR engagement. Among proteins whose phosphorylation was regulated by TCR stimulation, we demonstrated, using a fast‐track gene inactivation approach in primary lymphocytes, that the ITSN2 adaptor protein regulated T‐cell effector functions. This resource, called LymphoAtlas, represents an integrated pipeline to further decipher the organization of the signaling network encoding T‐cell activation. LymphoAtlas is accessible to the community at: https://bmm-lab.github.io/LymphoAtlas.

Clustering of the ζ-Chain Can Initiate T Cell Receptor Signaling

T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca2+ flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.

Kennedy pathway regulation by miR-16-1/15a cluster family during T cell activation

Abstract The Kennedy pathway in the endoplasmic reticulum is vital for the synthesis of phosphatidylethanolamine (PE) that is incorporated into cell membranes and lipid-based signaling molecules. The three steps of the Kennedy pathway are catalyzed by ethanolamine kinase, CTP-phosphoethanolamine Cytidyltransferase, and selenoprotein I (SELENOI). Our data suggest that two of the three enzymes (ethanolamine kinase and SELENOI) included in this pathway are upregulated during T cell activation as a mechanism for increasing PE, to support increased membrane synthesis and lipidated signaling proteins. Moreover, we have analyzed micro-RNAs (miRs) and found that miR-16-1 and miR-15a, which are linked in the genome and are co-expressed, have sequences predicted to target ethanolamine kinase and SELENOI. Thus, we hypothesized that levels of miR-16-1/15a decrease upon T cell activation to allow increased expression of ethanolamine kinase and SELENOI, thereby driving the increased synthesis of PE. Using real-time PCR and western blot analyses, we found that both miR-16-1 and miR-15a decreased, while levels of ethanolamine kinase and SELENOI increased in mouse primary T cells upon T cell receptor (TCR) activation. This coincided with increased levels of different PE species as measured by LC-MS. Overall, these data support the model of miR-16-1/15a regulation of the Kennedy pathway, in which miR-16-1/15a levels are higher in naïve T cells as a means to keep PE synthesis low. Upon TCR engagement, these miRs are reduced and all three enzymes are upregulated, thereby allowing increased PE synthesis required by activated T cells.

Antigen recognition by CD8+ T cells in non-lymphoid tissues initiates a transcriptional program of tissue-resident memory differentiation

Abstract Tissue-resident memory (TRM) CD8+ T cells permanently reside within non-lymphoid tissues to provide a first-line of defense against invading pathogens. TRM T cells are largely derived from recently activated effector T cells, but the mechanisms that control the extent of TRM differentiation once they are recruited into non-lymphoid tissue microenvironments are largely undefined. To determine how T cell receptor (TCR) engagement promotes TRM formation, we utilized interferon-gamma (IFNγ)-YFP reporter T cells and found that only ~25% of the antigen-specific CD8+ T cells in the skin produced IFNγ during the course of a Vaccinia virus (VacV) infection. Strikingly, expression of IFNγ was strictly antigen-dependent and genome-wide transcriptional profiling revealed that IFNγ+ CD8+ T cells in the VacV-infected skin were becoming tissue-residents, suggesting that local antigen recognition identifies the dominant TRM precursor population. Mechanistically, TCR engagement caused effector CD8+ T cells to express Blimp1 and repress expression of the tissue egress receptor S1PR1 and the transcription factor KLF2, collectively preventing S1P-dependent migration. Interestingly, the S1PR1 antagonist CD69 was not transcriptionally upregulated by TCR stimulation. However, CD69 surface expression was Blimp1 dependent, suggesting that Blimp1-mediated repression of S1PR1 causes increased availability of CD69 protein to remain on the cell surface, a hallmark of TRM differentiation. Thus, antigen recognition by effector CD8+ T cells in non-lymphoid tissues is a critical factor that promotes Blimp1 expression, ultimately causing stable expression of CD69 and retention of TRM CD8+ T cells in the skin following viral infection.

Molecular mechanisms underlying T cell co-potentiation by anti-CD3 Fab fragments

Abstract Cognate antigen binding to the T cell receptor (TCR) induces a conformational change of the CD3 complex (CD3Δc), which is required but not sufficient to trigger productive signaling from the TCR/CD3. CD3Δc fails to occur when the TCR ligates poorly immunogenic antigens. In the absence of CD3Δc, T cells do not display productive immune responses. CD3Δc involves the exposure of a conserved proline-rich-sequence (PRS) in CD3ɛ cytoplasmic tail that functions as a docking site for cytoplasmic adaptor proteins like Nck, which participate in the CD3 signaling cascade. The opening of the PRS is controlled by a CXXC motif in CD3ɛ located adjacent to the plasma membrane. We previously described it is possible to mimic CD3Δc on T cells when adding Fab fragments capable to bind to the CD3 complex. When combining TCR engagement to weak antigens with binding of Fabs to the CD3 complex that not interfere with antigen binding, T cell co-potentiation occurs. In this instance, T cells become capable to display responses to poorly immunogenic stimulation. As an example of the consequences of T cell co-potentiation in vivo, we studied anti-mouse CD3 Fabs as a novel immunotherapeutic against poorly immunogenic types of cancer. Anti-CD3 Fabs reduced melanoma burden in B6 mice bearing B16-F10 tumor in lungs. Now, we have explored the molecular mechanisms supporting T cell co-potentiation in monoclonal and polyclonal mouse models. In B6 mice expressing null mutations for either the CXXC or the PRS motifs, anti-CD3 Fabs fail to induce CD3Δc, as well as to co-potentiate murine T cells in vitro and in vivo. These data suggest PRS opening by anti-CD3 Fabs may be linked to their capacity to co-potentiate T cells responses to poorly immunogenic antigens.

Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters is controlled by IKKβ kinase activity

Abstract The IkB kinase (IKK) complex coordinates inflammatory responses by activating canonical NFkB downstream of immune receptors. This complex consists of the kinases IKKa/β and the dimeric adaptor subunit NEMO (NFkB essential modulator protein). NEMO mutations cause immunodeficiencies and impair antigen receptor function. Canonical NFkB activation by immunoreceptors, such as the T cell receptor (TCR), requires the assembly of the Carma1/Bcl10/Malt1 (CBM) signalosome. While, functional studies and in vitro interactions, place the IKK complex downstream of the CBM signalosome, the spatial relationship between these complexes has never been observed in intact immune cells. We observed that NEMO is recruited into TCR microclusters and membrane compartments within ~70 seconds of TCR engagement. Point mutations impacting the K63-linked and linear polyubiquitin ubiquitin-binding domains of NEMO selectively impair NEMO recruitment into TCR microclusters. We verified the involvement of these polyubiquitin chains by showing that inhibitors of either the K63-specific Uev1A/Ubc13 ubiquitin E2 ligase or the linear ubiquitin activating complex (LUBAC) prevent NEMO from entering TCR microclusters. To examine whether the entire IKK complex is recruited to the TCR, we co-expressed IKKb chimeras with NEMO. We observed that wild-type (WT), but not kinase-dead, IKKb caused NEMO microclusters to disappear. Similarly, the treatment of cells expressing WT IKKb with inhibitors of IKKβ or its upstream activator, TAK1, restored the recruitment of IKKβ and NEMO into microclusters. These results suggest that the IKK complex is recruited to the TCR via linear- and K63-linked polyubiquitin chains and then dissociates from the TCR via IKKβ activity.

C reactive protein impairs adaptive immunity in immune cells of patients with melanoma

BackgroundHigh C reactive protein (CRP) levels have been reported to be associated with a poor clinical outcome in a number of malignancies and with programmed cell death protein 1 immune...

The Role of Calcium-Calcineurin-NFAT Signaling Pathway in Health and Autoimmune Diseases.

Calcium (Ca2+) is an essential signaling molecule that controls a wide range of biological functions. In the immune system, calcium signals play a central role in a variety of cellular functions such as proliferation, differentiation, apoptosis, and numerous gene transcriptions. During an immune response, the engagement of T-cell and B-cell antigen receptors induces a decrease in the intracellular Ca2+ store and then activates store-operated Ca2+ entry (SOCE) to raise the intracellular Ca2+ concentration, which is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels. Recently, identification of the two critical regulators of the CRAC channel, stromal interaction molecule (STIM) and Orai1, has broadened our understanding of the regulatory mechanisms of Ca2+ signaling in lymphocytes. Repetitive or prolonged increase in intracellular Ca2+ is required for the calcineurin-mediated dephosphorylation of the nuclear factor of an activated T cell (NFAT). Recent data indicate that Ca2+-calcineurin-NFAT1 to 4 pathways are dysregulated in autoimmune diseases. Therefore, calcineurin inhibitors, cyclosporine and tacrolimus, have been used for the treatment of such autoimmune diseases as systemic lupus erythematosus and rheumatoid arthritis. Here, we review the role of the Ca2+-calcineurin–NFAT signaling pathway in health and diseases, focusing on the STIM and Orai1, and discuss the deregulated calcium-mediated calcineurin-NFAT pathway in autoimmune diseases.

Activation, Deficiency, and Reduced IFN-γ Production of Mucosal-Associated Invariant T Cells in Patients with Inflammatory Bowel Disease

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that can activate either in response to T-cell receptor (TCR) engagement or through activating cytokines and play an important role in autoimmune disorders. The study examined the level and function of MAIT cells in patients with inflammatory bowel disease (IBD). Circulating MAIT cell levels were significantly reduced in IBD patients. This MAIT cell deficiency was correlated with IBD disease activity grades, hemoglobin, and CRP. IFN-γ production of circulating MAIT cells in response to both MHC class 1b-like related protein (MR1)-dependent and -independent stimulations was decreased in IBD patients, which was partially associated with reduced activation of nuclear factor of activated T cells 1 (NFAT1) transcription factor, a main regulator of IFN-γ production. Expression levels of CD69, programmed death-1 (PD-1), and annexin V in MAIT cells were elevated in IBD patients. CCL20, CXCL10, CXCL16, and CCL25 were expressed higher in inflamed intestinal tissues than in noninflamed tissues. This study demonstrates that circulating MAIT cells are activated and numerically and functionally deficient in IBD patients. Furthermore, activated MAIT cells have the potential to migrate to inflamed tissues. These findings suggest an important role of MAIT cells in mucosal immunity in IBD.

The structural basis of T-cell receptor (TCR) activation: An enduring enigma.

T cells are critical for protective immune responses to pathogens and tumors. The T-cell receptor (TCR)-CD3 complex is composed of a diverse αβ TCR heterodimer noncovalently associated with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates recognition of antigenic peptides bound to MHC molecules (pMHC), whereas the CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T-cell signaling pathways, the mechanism whereby TCR engagement by pMHC is first communicated to the CD3 signaling apparatus, a process termed early T-cell activation, remains largely a mystery. In this review, we examine the molecular basis for TCR activation in light of the recently determined cryoEM structure of a complete TCR-CD3 complex. This structure provides an unprecedented opportunity to assess various signaling models that have been proposed for the TCR. We review evidence from single-molecule and structural studies for force-induced conformational changes in the TCR-CD3 complex, for dynamically-driven TCR allostery, and for pMHC-induced structural changes in the transmembrane and cytoplasmic regions of CD3 subunits. We identify major knowledge gaps that must be filled in order to arrive at a comprehensive model of TCR activation that explains, at the molecular level, how pMHC-specific information is transmitted across the T-cell membrane to initiate intracellular signaling. An in-depth understanding of this process will accelerate the rational design of immunotherapeutic agents targeting the TCR-CD3 complex.

CD8+ T Lymphocyte and NK Cell Network: Circuitry in the Cytotoxic Domain of Immunity.

Multiple effector layers in the immune system ensure an optimal temporal and spatial distribution of immune defense. Cytotoxic innate lymphoid natural killers (NK) and adaptive CD8+ T lymphocytes (CTL) interact to elicit specific cytolytic outcomes. The CTL carry antigen-specific T cell receptors (TCR) to recognize cognate peptides bound with major histocompatibility complex class-I (MHC-I) or human leukocyte antigen (HLA) molecules on target cells. Upon TCR engagement with MHC-I:peptide at a threshold of avidity, T cell intracellular programs converge into cytolytic activity. By contrast, NK cells lack antigen-specific receptors but express a repertoire of highly polymorphic and polygenic inhibitory and activating receptors that bind various ligands including MHC and like molecules. A highly calibrated maturation enables NK cells to eliminate target cells with lowered or absent MHC-I or induced MHC-I-related molecules while maintaining their tolerance toward self-MHC. Both CTL and mature NK cells undergo membranous reorganization and express various effector molecules to eliminate aberrant cells undergoing a stress of transformation, infection or other pathological noxa. Here, we present the cellular modules that underlie the CTL–NK circuitry to maximize their effector cooperativity against stressed or cancerous cells.

Abstract 2696: Genetic and pharmacologic evaluation of the ubiquitin ligase CBL-B as a small-molecule, tumor immunotherapy target

Abstract E3 ubiquitin ligases play critical roles in directing cellular protein fate by controlling the specificity of ubiquitin conjugation to substrate proteins and targeting them for cellular relocalization or degradation by the ubiquitin proteasome system. The E3 ubiquitin ligase CBL-B is expressed in immune cell lineages and negatively regulates activity of the T-cell receptor (TCR) by imposing a requirement for a costimulatory signal to mount a productive immune response upon TCR engagement. Mice deficient in Cbl-b, and more specifically in the RING Zn-finger ligase domain of Cbl-b, demonstrate a tumor rejection phenotype mediated by CD8+ T cells (Paolino et al., JI, 2011). We have reproduced these results and demonstrate that Cbl-b deficient mice show enhanced anti-tumor activity. In addition, we show that CD4+ and CD8+ T cells from mice deficient in Cbl-b have 5 to 10-fold enhanced secretion of IL-2 and IFN γ when stimulated ex vivo. These data provide a genetic rationale for the development of a small molecule inhibitor of CBL-B ligase activity for use in patients with tumor-mediated immune suppression of effector T cells. We have identified a series of small molecule inhibitors of CBL-B activity with biochemical potency at low nanomolar concentrations. CBL-B inhibitors increased cytokine secretion in vitro at low nanomolar concentrations, as measured by IL-2 and IFN γ secretion, in primary human and mouse T cells stimulated with CD3/CD28 or CD3 alone. The compounds also stimulated proliferation and elevated levels of the T cell surface activation markers CD25 and CD69. CBL-B inhibitors enhanced an antigen recall response in human PBMCs ex vivo, as measured by approximately 5-fold higher secretion of GM-CSF, TNF-α and RANTES, and demonstrated effects in an ex vivo model of exhausted T cell function. Oral dosing of an optimized CBL-B inhibitor enhanced anti-CD3 stimulated T cell activation in mouse CD4+ and CD8+ T cells, demonstrating a dose proportional pharmacodynamic effect. Oral administration over 28 days in the syngeneic CT-26 tumor model was well tolerated and resulted in single agent tumor growth inhibition. These data support the continued advancement of small molecule oral CBL-B inhibitors for future development in immuno-oncology. Citation Format: Jennifa Gosling, Ryan Rountree, Asad Taherbhoy, Chenbo Wang, Thomas Cummins, Frederick Cohen, Hiroko Tanaka, Dahlia Weiss, Mario Cardozo, Christopher Karim, May Tan, Joseph Juan, Austin Tenn-McClellan, Szerenke Kiss von Soly, Julie Sheung, Kathleen Boyle, Ketki Dhamnaskar, Katherine Kurylo, Jilliane Bruffey, Jennifer McKinnell, Dane Karr, Andria Christianson, Anne-Renee Van Der Vuurst de Vries, Pallavur Sivakumar, Mark Gallop, Paul A. Barsanti, Anjanabha Saha, Neil F. Bence, Christoph W. Zapf. Genetic and pharmacologic evaluation of the ubiquitin ligase CBL-B as a small-molecule, tumor immunotherapy target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2696.

The Role of Adaptor Proteins in the Biology of Natural Killer T (NKT) Cells

Adaptor proteins contribute to the selection, differentiation and activation of natural killer T (NKT) cells, an innate(-like) lymphocyte population endowed with powerful immunomodulatory properties. Distinct from conventional T lymphocytes NKT cells preferentially home to the liver, undergo a thymic maturation and differentiation process and recognize glycolipid antigens presented by the MHC class I-like molecule CD1d on antigen presenting cells. NKT cells express a semi-invariant T cell receptor (TCR), which combines the Vα14-Jα18 chain with a Vβ2, Vβ7, or Vβ8 chain in mice and the Vα24 chain with the Vβ11 chain in humans. The avidity of interactions between their TCR, the presented glycolipid antigen and CD1d govern the selection and differentiation of NKT cells. Compared to TCR ligation on conventional T cells engagement of the NKT cell TCR delivers substantially stronger signals, which trigger the unique NKT cell developmental program. Furthermore, NKT cells express a panoply of primarily inhibitory NK cell receptors (NKRs) that control their self-reactivity and avoid autoimmune activation. Adaptor proteins influence NKT cell biology through the integration of TCR, NKR and/or SLAM (signaling lymphocyte-activation molecule) receptor signals or the variation of CD1d-restricted antigen presentation. TCR and NKR ligation engage the SH2 domain-containing leukocyte protein of 76kDa slp-76 whereas the SLAM associated protein SAP serves as adaptor for the SLAM receptor family. Indeed, the selection and differentiation of NKT cells selectively requires co-stimulation via SLAM receptors. Furthermore, SAP deficiency causes X-linked lymphoproliferative disease with multiple immune defects including a lack of circulating NKT cells. While a deletion of slp-76 leads to a complete loss of all peripheral T cell populations, mutations in the SH2 domain of slp-76 selectively affect NKT cell biology. Furthermore, adaptor proteins influence the expression and trafficking of CD1d in antigen presenting cells and subsequently selection and activation of NKT cells. Adaptor protein complex 3 (AP-3), for example, is required for the efficient presentation of glycolipid antigens which require internalization and processing. Thus, our review will focus on the complex contribution of adaptor proteins to the delivery of TCR, NKR and SLAM receptor signals in the unique biology of NKT cells and CD1d-restricted antigen presentation.