Antibody agonists trigger immune receptor signaling through local exclusion of receptor-type protein tyrosine phosphatases.

Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.

Dual role of the peptide-loading complex as proofreader and limiter of MHC-I presentation.

Antigen presentation via major histocompatibility complex class I (MHC-I) molecules is essential for surveillance by the adaptive immune system. Central to this process is the peptide-loading complex (PLC), which translocates peptides from the cytosol to the endoplasmic reticulum and catalyzes peptide loading and proofreading of peptide-MHC-I (pMHC-I) complexes. Despite its importance, the impact of individual PLC components on the presented pMHC-I complexes is still insufficiently understood. Here, we used stoichiometrically defined antibody-nanobody complexes and engineered soluble T cell receptors (sTCRs) to quantify different MHC-I allomorphs and defined pMHC-I complexes, respectively. Thereby, we uncovered distinct effects of individual PLC components on the pMHC-I surface pool. Knockouts of components of the PLC editing modules, namely tapasin, ERp57, or calreticulin, changed the MHC-I surface composition to a reduced proportion of HLA-A*02:01 presentation compensated by a higher ratio of HLA-B*40:01 molecules. Intriguingly, these knockouts not only increased the presentation of suboptimally loaded HLA-A*02:01 complexes but also elevated the presentation of high-affinity peptides overexpressed in the cytosol. Our findings suggest that the components of the PLC editing module serve a dual role, acting not only as peptide proofreaders but also as limiters for abundant peptides. This dual function ensures the presentation of a broad spectrum of antigenic peptides.

Inhibitory CARs fail to protect from immediate T cell cytotoxicity.

Chimeric antigen receptors (CARs) equipped with an inhibitory signaling domain (iCARs) have been proposed as strategy to increase on-tumor specificity of CAR-T cell therapies. iCARs inhibit T cell activation upon antigen recognition and thereby program a Boolean NOT gate within the CAR-T cell. If cancer cells do not express the iCAR target antigen while it is highly expressed on healthy tissue, CAR/iCAR coexpressing T cells are supposed to kill cancer cells but not healthy cells expressing the CAR antigen. In this study, we employed a well-established reporter cell system to demonstrate high potency of iCAR constructs harboring BTLA-derived signaling domains. We then created CAR/iCAR combinations for the clinically relevant antigen pairs B7-H3/CD45 and CD123/CD19 and show potent reporter cell suppression by iCARs targeting CD45 or CD19. In primary human T cells αCD19-iCARs were capable of suppressing T cell proliferation and cytokine production. Surprisingly, the iCAR failed to veto immediate CAR-mediated cytotoxicity. Likewise, T cells overexpressing PD-1 or BTLA did not show impaired cytotoxicity toward ligand-expressing target cells, indicating that inhibitory signaling by these receptors does not mediate protection against cytotoxicity by CAR-T cells. Future approaches employing iCAR-equipped CAR-T cells for cancer therapy should therefore monitor off-tumor reactivity and potential CAR/iCAR-T cell dysfunction.

Plant-derived Durvalumab variants show efficient PD-1/PD-L1 blockade and therapeutically favourable FcR binding.

Immune checkpoint blocking therapy targeting the PD-1/PD-L1 inhibitory signalling pathway has produced encouraging results in the treatment of a variety of cancers. Durvalumab (Imfinzi®) targeting PD-L1 is currently used for immunotherapy of several tumour malignancies. The Fc region of this IgG1 antibody has been engineered to reduce FcγR interactions with the aim of enhancing blockade of PD-1/PD-L1 interactions without the depletion of PD-L1-expressing immune cells. Here, we used Nicotiana benthamiana to produce four variants of Durvalumab (DL): wild-type IgG1 and its 'Fc-effector-silent' variant (LALAPG) carrying further modifications to increase antibody half-life (YTE); IgG4S228P and its variant (PVA) with Fc mutations to decrease binding to FcγRI. In addition, DL variants were produced with two distinct glycosylation profiles: afucosylated and decorated with α1,6-core fucose. Plant-derived DL variants were compared to the therapeutic antibody regarding their ability to (i) bind to PD-L1, (ii) block PD-1/PD-L1 inhibitory signalling and (iii) engage with the neonatal Fc receptor (FcRn) and various Fcγ receptors. It was found that plant-derived DL variants bind to recombinant PD-L1 and to PD-L1 expressed in gastrointestinal cancer cells and are able to effectively block its interaction with PD-1 on T cells, thereby enhancing their activation. Furthermore, we show a positive impact of Fc amino acid mutations and core fucosylation on DL's therapeutic potential. Compared to Imfinzi®, DL-IgG1 (LALAPG) and DL-IgG4 (PVA)S228P show lower affinity to CD32B inhibitory receptor which can be therapeutically favourable. Importantly, DL-IgG1 (LALAPG) also shows enhanced binding to FcRn, a key determinant of serum half-life of IgGs.

The CD6 interactome orchestrates ligand-independent T cell inhibitory signaling.

BACKGROUND: T-cell membrane scaffold proteins are pivotal in T cell function, acting as versatile signaling hubs. While CD6 forms a large intracellular signalosome, it is distinguished from typical scaffolds like LAT or PAG by possessing a substantial ectodomain that binds CD166, a well-characterized ligand expressed on most antigen-presenting cells (APC), through the third domain (d3) of the extracellular region. Although the intact form of CD6 is the most abundant in T cells, an isoform lacking d3 (CD6∆d3) is transiently expressed on activated T cells. Still, the precise character of the signaling transduced by CD6, whether costimulatory or inhibitory, and the influence of its ectodomain on these activities are unclear. METHODS: We expressed CD6 variants with extracellular deletions or cytosolic mutations in Jurkat cells containing eGFP reporters for NF-κB and NF-AT transcription factor activation. Cell activation was assessed by eGFP flow cytometry following Jurkat cell engagement with superantigen-presenting Raji cells. Using imaging flow cytometry, we evaluated the impact of the CD6-CD166 pair on cell adhesiveness during the antigen-dependent and -independent priming of T cells. We also examined the role of extracellular or cytosolic sequences on CD6 translocation to the immunological synapse, using immunofluorescence-based imaging. RESULTS: Our investigation dissecting the functions of the extracellular and cytosolic regions of CD6 revealed that CD6 was trafficked to the immunological synapse and exerted tonic inhibition wholly dependent on its cytosolic tail. Surprisingly, however, translocation to the synapse occurred independently of the extracellular d3 and of engagement to CD166. On the other hand, CD6 binding to CD166 significantly increased T cell:APC adhesion. However, this activity was most evident in the absence of APC priming with superantigen, and thus, in the absence of TCR engagement. CONCLUSIONS: Our study identifies CD6 as a novel 'on/off' scaffold-receptor capable of modulating responsiveness in two ways. Firstly, and independently of ligand binding, it establishes signaling thresholds through tonic inhibition, functioning as a membrane-bound scaffold. Secondly, CD6 has the capacity for alternative splicing-dependent variable ligand engagement, modulating its checkpoint-like activity.

Engineered soluble, trimerized 4-1BBL variants as potent immunomodulatory agents.

Targeting co-stimulatory receptors promotes the activation and effector functions of anti-tumor lymphocytes. 4-1BB (CD137/TNFSF9), a member of the tumor necrosis factor receptor superfamily (TNFR-SF), is a potent co-stimulatory receptor that plays a prominent role in augmenting effector functions of CD8+ T cells, but also CD4+ T cells and NK cells. Agonistic antibodies against 4-1BB have entered clinical trials and shown signs of therapeutic efficacy. Here, we have used a T cell reporter system to evaluate various formats of 4-1BBL regarding their capacity to functionally engage its receptor. We found that a secreted 4-1BBL ectodomain harboring a trimerization domain derived from human collagen (s4-1BBL-TriXVIII) is a strong inducer of 4-1BB co-stimulation. Similar to the 4-1BB agonistic antibody urelumab, s4-1BBL-TriXVIII is very potent in inducing CD8+ and CD4+ T cell proliferation. We provide first evidence that s4-1BBL-TriXVIII can be used as an effective immunomodulatory payload in therapeutic viral vectors. Oncolytic measles viruses encoding s4-1BBL-TriXVIII significantly reduced tumor burden in a CD34+ humanized mouse model, whereas measles viruses lacking s4-1BBL-TriXVIII were not effective. Natural soluble 4-1BB ligand harboring a trimerization domain might have utility in tumor therapy especially when delivered to tumor tissue as systemic administration might induce liver toxicity.

A conformation-specific ON-switch for controlling CAR T cells with an orally available drug.

Molecular ON-switches in which a chemical compound induces protein-protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.

Human repair-related Schwann cells adopt functions of antigen-presenting cells in vitro.

The plastic potential of Schwann cells (SCs) is increasingly recognized to play a role after nerve injury and in diseases of the peripheral nervous system. Reports on the interaction between immune cells and SCs indicate their involvement in inflammatory processes. However, the immunocompetence of human SCs has been primarily deduced from neuropathies, but whether after nerve injury SCs directly regulate an adaptive immune response is unknown. Here, we performed comprehensive analysis of immunomodulatory capacities of human repair-related SCs (hrSCs), which recapitulate SC response to nerve injury in vitro. We used our well-established culture model of primary hrSCs from human peripheral nerves and analyzed the transcriptome, secretome, and cell surface proteins for pathways and markers relevant in innate and adaptive immunity, performed phagocytosis assays, and monitored T-cell subset activation in allogeneic co-cultures. Our findings show that hrSCs are phagocytic, which is in line with high MHCII expression. Furthermore, hrSCs express co-regulatory proteins, such as CD40, CD80, B7H3, CD58, CD86, and HVEM, release a plethora of chemoattractants, matrix remodeling proteins and pro- as well as anti-inflammatory cytokines, and upregulate the T-cell inhibiting PD-L1 molecule upon pro-inflammatory stimulation with IFNγ. In contrast to monocytes, hrSC alone are not sufficient to trigger allogenic CD4+ and CD8+ T-cells, but limit number and activation status of exogenously activated T-cells. This study demonstrates that hrSCs possess features and functions typical for professional antigen-presenting cells in vitro, and suggest a new role of these cells as negative regulators of T-cell immunity during nerve regeneration.

NKG2A-checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA-E.

NKG2A has emerged as a new immunotherapy target and its blockade with the novel immune checkpoint inhibitor (ICI) monalizumab can boost both NK cell and CD8+ T cell responses. NKG2A forms heterodimers with CD94 and binds to the human non-classical MHC class I molecule HLA-E. HLA-E forms complexes with a limited set of peptides mainly derived from the leader sequences of the classical MHC class I molecules (HLA-A, HLA-B and HLA-C) and the non-classical class I paralogue HLA-G, and it is well established that the interaction between CD94/NKG2x receptors and its ligand HLA-E is peptide-sensitive. Here, we have evaluated peptide dependence of NKG2A-mediated inhibition and the efficiency of interference by monalizumab in a transcriptional T cell reporter system. NKG2A inhibition was mediated by cell-expressed HLA-E molecules stably presenting disulfate-trapped peptide ligands. We show that different HLA-class I leader peptides mediate varying levels of inhibition. We have used NKG2A/NKG2C chimeric receptors to map the binding site of NKG2A and NKG2C blocking antibodies. Furthermore, we determined the functional EC50 values of blocking NKG2A antibodies and show that they greatly depend on the HLA-leader peptide presented by HLA-E. Monalizumab was less effective in augmenting NK cell-mediated killing of target cells displaying HLA-G peptide on HLA-E, than cells expressing HLA-E complexed with HLA-A, HLA-B and HLA-C peptides. Our results indicate that peptides displayed by HLA-E molecules on tumour cells might influence the effectivity of NKG2A-ICI therapy and potentially suggest novel approaches for patient stratification, for example, based on tumoral HLA-G levels.

PD-1 blocking antibodies moonlighting as killers.

Therapeutic antibodies that block PD-1-mediated inhibition of T cells have revolutionized cancer therapy. Murine cancer models are an essential tool for testing the efficacy of PD-1 blockers alone or in combination with other treatments. Depending on the isotype of the antibody and the host species, blocking antibodies can also exert cytotoxic activity towards cells expressing the target molecule. In the current issue of the European Journal of Immunology [Eur. J. Immunol. 2021. 51: 1473-1481], Polesso et al. demonstrate that depletion of PD-1+ T cells by "blocking" PD-1 antibodies can greatly impact the outcome of preclinical immunotherapy experiments. Whereas some PD-1 antibodies promoted activation and proliferation of PD-1-expressing murine T cells, the authors report that administration of a particular PD-1 antibody can result in a significant loss of antigen-specific CD8 T cells in different in vivo models. These findings once more highlight that a comprehensive characterization of antibodies is warranted to avoid misinterpretation of immunotherapy studies.

4-1BB costimulation promotes bystander activation of human CD8 T cells.

Costimulatory signals potently promote T-cell proliferation and effector function. Agonistic antibodies targeting costimulatory receptors of the TNFR family, such as 4-1BB and CD27, have entered clinical trials in cancer patients. Currently there is limited information how costimulatory signals regulate antigen-specific but also bystander activation of human CD8 T cells. Engineered antigen presenting cells (eAPC) efficiently presenting several common viral epitopes on HLA-A2 in combination with MHC class I tetramer staining were used to investigate the impact of costimulatory signals on human CD8 T-cell responses. CD28 costimulation potently augmented the percentage and number of antigen-reactive CD8 T cells, whereas eAPC expressing 4-1BB-ligand induced bystander proliferation of CD8 T cells and massive expansion of NK cells. Moreover, the 4-1BB agonist urelumab similarly induced bystander proliferation of CD8 T cells and NK cells in a dose-dependent manner. However, the promotion of bystander CD8 T-cell responses is not a general attribute of costimulatory TNF receptor superfamily (TNFRSF) members, since CD27 signals enhanced antigen-specific CD8 T cells responses without promoting significant bystander activation. Thus, the differential effects of costimulatory signals on the activation of human bystander CD8 T cells should be taken into account when costimulatory pathways are harnessed for cancer immunotherapy.

Differentiation and activation of human CD4 T cells is associated with a gradual loss of myelin and lymphocyte protein.

Upon generation of monoclonal antibodies to the T cell antigen receptor/CD3 (TCR/CD3) complex, we isolated mAb MT3, whose reactivity correlates inversely with the production of IFN-γ by human peripheral blood T lymphocytes. Using eukaryotic expression cloning, we identified the MT3 antigen as myelin-and-lymphocyte (MAL) protein. Flow cytometry analysis demonstrates high surface expression of MAL on all naïve CD4+ T cells whereas MAL expression is diminished on central memory- and almost lost on effector memory T cells. MAL- T cells proliferate strongly in response to stimulation with CD3/CD28 antibodies, corroborating that MAL+ T cells are naïve and MAL- T cells memory subtypes. Further, resting MAL- T cells harbor a larger pool of Ser59- and Tyr394- double phosphorylated lymphocyte-specific kinase (Lck), which is rapidly increased upon in vitro restimulation. Previously, lack of MAL was reported to prevent transport of Lck, the key protein tyrosine kinase of TCR/CD3 signaling to the cell membrane, and to result in strongly impaired human T cell activation. Here, we show that knocking out MAL did not significantly affect Lck membrane localization and immune synapse recruitment, or transcriptional T cell activation. Collectively, our results indicate that loss of MAL is associated with activation-induced differentiation of human T cells but not with impaired membrane localization of Lck or TCR signaling capacity.

The soluble cytoplasmic tail of CD45 regulates T-cell activation via TLR4 signaling.

The soluble cytoplasmic tail of CD45 (ct-CD45) is a cleavage fragment of CD45, that is generated during the activation of human phagocytes. Upon release to the extracellular space, ct-CD45 binds to human T cells and inhibits their activation in vitro. Here, we studied the potential role of TLR4 as a receptor for ct-CD45. Treatment of Jurkat TLR4/CD14 reporter cells with ct-CD45 induced the upregulation of the reporter gene NFκB-eGFP and could be blocked by inhibitors of TLR4 signaling. Conversely, ct-CD45 did not promote the NFκB-controlled eGFP induction in reporter cells expressing TLR1, TLR2, and TLR6 transgenes and did not lead to the activation of the transcription factors NFκB, AP-1, and NFAT in a Jurkat reporter cell line expressing endogenous TLR5. Moreover, ct-CD45 binds to recombinant TLR4 in an in vitro assay and this association was reduced in the presence of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine. Blockade of TLR4 with mAb HTA125 partially reversed the ct-CD45-mediated inhibition of T-cell proliferation. Interestingly, targeting of TLR4 with mAb W7C11 also suppressed T-cell proliferation. In summary, the results of this study demonstrate that ct-CD45 acts via a noncanonical TLR4 activation pathway on T cells, which modulates TCR signaling.

Saturated very long-chain fatty acids regulate macrophage plasticity and invasiveness.

Saturated very long-chain fatty acids (VLCFA, ≥ C22), enriched in brain myelin and innate immune cells, accumulate in X-linked adrenoleukodystrophy (X-ALD) due to inherited dysfunction of the peroxisomal VLCFA transporter ABCD1. In its severest form, X-ALD causes cerebral myelin destruction with infiltration of pro-inflammatory skewed monocytes/macrophages. How VLCFA levels relate to macrophage activation is unclear. Here, whole transcriptome sequencing of X-ALD macrophages indicated that VLCFAs prime human macrophage membranes for inflammation and increased expression of factors involved in chemotaxis and invasion. When added externally to mimic lipid release in demyelinating X-ALD lesions, VLCFAs did not activate toll-like receptors in primary macrophages. In contrast, VLCFAs provoked pro-inflammatory responses through scavenger receptor CD36-mediated uptake, cumulating in JNK signalling and expression of matrix-degrading enzymes and chemokine release. Following pro-inflammatory LPS activation, VLCFA levels increased also in healthy macrophages. With the onset of the resolution, VLCFAs were rapidly cleared in control macrophages by increased peroxisomal VLCFA degradation through liver-X-receptor mediated upregulation of ABCD1. ABCD1 deficiency impaired VLCFA homeostasis and prolonged pro-inflammatory gene expression upon LPS treatment. Our study uncovers a pivotal role for ABCD1, a protein linked to neuroinflammation, and associated peroxisomal VLCFA degradation in regulating macrophage plasticity.

Integrated drug profiling and CRISPR screening identify essential pathways for CAR T-cell cytotoxicity.

Chimeric antigen receptor (CAR) T-cell therapy has proven effective in relapsed and refractory B-cell malignancies, but resistance and relapses still occur. Better understanding of mechanisms influencing CAR T-cell cytotoxicity and the potential for modulation using small-molecule drugs could improve current immunotherapies. Here, we systematically investigated druggable mechanisms of CAR T-cell cytotoxicity using >500 small-molecule drugs and genome-scale CRISPR-Cas9 loss-of-function screens. We identified several tyrosine kinase inhibitors that inhibit CAR T-cell cytotoxicity by impairing T-cell signaling transcriptional activity. In contrast, the apoptotic modulator drugs SMAC mimetics sensitized B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma cells to anti-CD19 CAR T cells. CRISPR screens identified death receptor signaling through FADD and TNFRSF10B (TRAIL-R2) as a key mediator of CAR T-cell cytotoxicity and elucidated the RIPK1-dependent mechanism of sensitization by SMAC mimetics. Death receptor expression varied across genetic subtypes of B-cell malignancies, suggesting a link between mechanisms of CAR T-cell cytotoxicity and cancer genetics. These results implicate death receptor signaling as an important mediator of cancer cell sensitivity to CAR T-cell cytotoxicity, with potential for pharmacological targeting to enhance cancer immunotherapy. The screening data provide a resource of immunomodulatory properties of cancer drugs and genetic mechanisms influencing CAR T-cell cytotoxicity.

Targeting the HVEM protein using a fragment of glycoprotein D to inhibit formation of the BTLA/HVEM complex.

Cancer immunotherapy using blockade of immune checkpoints is mainly based on monoclonal antibodies. Despite the tremendous success achieved by using those molecules to block immune checkpoint proteins, antibodies possess some weaknesses, which means that there is still a need to search for new compounds as alternatives to antibodies. Many current approaches are focused on use of peptides/peptidomimetics to destroy receptor/ligand interactions. Our studies concern blockade of the BTLA/HVEM complex, which generates an inhibitory effect on the immune response resulting in tolerance to cancer cells. To design inhibitors of such proteins binding we based our work on the amino acid sequence and structure of a ligand of HVEM protein, namely glycoprotein D, which possesses the same binding site on HVEM as BTLA protein. To disrupt the BTLA and HVEM interaction we designed several peptides, all fragments of glycoprotein D, and tested their binding to HVEM using SPR and their ability to inhibit the BTLA/HVEM complex formation using ELISA tests and cellular reporter platforms. That led to identification of two peptides, namely gD(1-36)(K10C-D30C) and gD(1-36)(A12C-L25C), which interact with HVEM and possess blocking capacities. Both peptides are not cytotoxic to human PBMCs, and show stability in human plasma. We also studied the 3D structure of the gD(1-36)(K10C-D30C) peptide using NMR and molecular modeling methods. The obtained data reveal that it possesses an unstructured conformation and binds to HVEM in the same location as gD and BTLA. All these results suggest that peptides based on the binding fragment of gD protein represent promising immunomodulation agents for future cancer immunotherapy.

Design, synthesis and biological evaluation of PD-1 derived peptides as inhibitors of PD-1/PD-L1 complex formation for cancer therapy.

Over the past few years, many molecules such as monoclonal antibodies, affibodies, nanobodies, and small compounds have been designed and tested as inhibitors of PD-1/PD-L1 complex formation. Some of them have been successfully implemented into clinical oncology practice. However, the majority of these compounds have disadvantages and limitations, such as high production price, potential for immunogenicity and/or prolonged clearance. Thus, new inhibitors of the PD-1/PD-L1 immune checkpoints are needed. Recently, peptides emerged as potential novel approach for blocking receptor/ligand interaction. In the presented studies we have designed, synthesised and tested peptides, which are potential inhibitors of the PD-1/PD-L1 axis. The amino acid sequences of the designed peptides were based on the binding sites of PD-1 to PD-L1, as determined by the crystal structure of the protein complex and also based on MM/GBSA analysis. Interactions of the peptides with PD-L1 protein were confirmed using SPR, while their inhibitory properties were studied using cell-based PD-1/PD-L1 immune checkpoint blockade assays. The characterization of the peptides has shown that the peptides PD-1(119-142)T120C-E141C, PD-1(119-142)C123-S137C and PD-1(122-138)C123-S137C strongly bind to PD-L1 protein and disrupt the interaction of the proteins. PD-1(122-138)C123-S137C peptide was shown to have the best inhibitory potential from the panel of peptides. Its 3D NMR structure was determined and the binding site to PD-L1 was established using molecular modelling methods. Our results indicate that the PD-1 derived peptides are able to mimic the PD-1 protein and inhibit PD-1/PD-L1 complex formation.

Composite CD79A/CD40 co-stimulatory endodomain enhances CD19CAR-T cell proliferation and survival.

Adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have led to impressive clinical outcomes in B cell malignancies. Beyond induction of remission, the persistence of CAR-T cells is required to prevent relapse and provide long-term disease control. To improve CAR-T cell function and persistence, we developed a composite co-stimulatory domain of a B cell signaling moiety, CD79A/CD40, to induce a nuclear translocating signal, NF-κB, to synergize with other T cell signals and improve CAR-T cell function. CD79A/CD40 incorporating CD19CAR-T cells (CD19.79a.40z) exhibited higher NF-κB and p38 activity upon CD19 antigen exposure compared with the CD28 or 4-1BB incorporating CD19CAR-T cells (CD19.28z and CD19.BBz). Notably, we found that CD19.79a.40z CAR-T cells continued to suppress CD19+ target cells throughout the co-culture assay, whereas a tendency for tumor growth was observed with CD19.28z CAR-T cells. Moreover, CD19.79a.40z CAR-T cells exhibited robust T cell proliferation after culturing with CD19+ target cells, regardless of exogenous interleukin-2. In terms of in vivo efficiency, CD19.79a.40z demonstrated superior anti-tumor activity and in vivo CAR-T cell proliferation compared with CD19.28z and CD19.BBz CD19CAR-T cells in Raji-inoculated mice. Our data demonstrate that the CD79A/CD40 co-stimulatory domain endows CAR-T cells with enhanced proliferative capacity and improved anti-tumor efficacy in a murine model.

TIM-3 and CEACAM1 do not interact in cis and in trans.

TIM-3 has been considered as a target in cancer immunotherapy. In T cells, inhibitory as well as activating functions have been ascribed to this molecule. Its role may therefore depend on the state of T cells and on the presence of interaction partners capable to perform functional pairing. Carcinoembryonic antigen-related cell adhesion molecule (CEACAM1) has been proposed to bind TIM-3 and to regulate its function. Using a T cell reporter platform we confirmed CEACAM1-mediated inhibition, but CEACAM1 did not functionally engage TIM-3. TIM-3 and CEACAM1 coexpression was limited to a small subset of activated T cells. Moreover, results obtained in extensive binding studies were not in support of an interaction between TIM-3 and CEACAM1. Cytoplasmic sequences derived from TIM-3 induced inhibitory signaling in our human T cell reporter system. Our results indicate that TIM-3 functions are independent of CEACAM1 and that this receptor has the capability to promote inhibitory signaling pathways in human T cells.

Diacylglycerol kinase α inhibition cooperates with PD-1-targeted therapies to restore the T cell activation program.

BACKGROUND: Antibody-based therapies blocking the programmed cell death-1/ligand-1 (PD-1/PD-L1) axis have provided unprecedent clinical success in cancer treatment. Acquired resistance, however, frequently occurs, commonly associated with the upregulation of additional inhibitory molecules. Diacylglycerol kinase (DGK) α limits the extent of Ras activation in response to antigen recognition, and its upregulation facilitates hypofunctional, exhausted T cell states. Pharmacological DGKα targeting restores cytotoxic function of chimeric antigen receptor and CD8+ T cells isolated from solid tumors, suggesting a mechanism to reverse T cell exhausted phenotypes. Nevertheless, the contribution of DGKα downstream of the PD-1/PD-L1 inhibitory axis in human T cells and the consequences of combining DGKα and anti-PD-1/PD-L1 inhibitors are still unresolved relevant issues. MATERIALS AND METHODS: We used a human triple parameter reporter cell line to investigate DGKα contribution to the PD-1/PD-L1 inhibitory pathway. We also addressed the impact of deleting DGKα expression in the growth dynamics and systemic tumor-derived effects of a PD-1-related tumor model, the MC38 colon adenocarcinoma. RESULTS: We identify DGKα as a contributor to the PD-1/PD-L1 axis that strongly limits the Ras/ERK/AP-1 pathway. DGKα function reinforces exhausted T cell phenotypes ultimately promoting tumor growth and generalized immunosuppression. Pharmacological DGKα inhibition selectively enhances AP-1 transcription and, importantly, cooperates with antibodies blocking the PD-1/PD-L1 interrelation. CONCLUSIONS: Our results indicate that DGKα inhibition could provide an important mechanism to revert exhausted T lymphocyte phenotypes and thus favor proper anti-tumor T cell responses. The cooperative effect observed after PD-1/PD-L1 and DGKα blockade offers a promising strategy to improve the efficacy of immunotherapy in the treatment of cancer.