BTLA and PD-1 signals attenuate TCR-mediated transcriptomic changes.

T cell co-inhibitory immune checkpoints, such as PD-1 or BTLA, are bona fide targets in cancer therapy. We used a human T cell reporter line to measure transcriptomic changes mediated by PD-1- and BTLA-induced signaling. T cell receptor (TCR)-complex stimulation resulted in the upregulation of a large number of genes but also in repression of a similar number of genes. PD-1 and BTLA signals attenuated transcriptomic changes mediated by TCR-complex signaling: upregulated genes tended to be suppressed and the expression of a significant number of downregulated genes was higher during PD-1 or BTLA signaling. BTLA was a significantly stronger attenuator of TCR-complex-induced transcriptome changes than PD-1. A strong overlap between genes that were regulated indicated quantitative rather than qualitative differences between these receptors. In line with their function as attenuators of TCR-complex-mediated changes, we found strongly regulated genes to be prime targets of PD-1 and BTLA signaling.

Co-stimulation of CD28/CD40 signaling molecule potentiates CAR-T cell efficacy and stemness.

CD19 chimeric antigen receptor T (CD19CAR-T) cells have achieved promising outcomes in relapsed/refractory B cell malignancies. However, recurrences occur due to the loss of CAR-T cell persistence. We developed dual T/B cell co-stimulatory molecules (CD28 and CD40) in CAR-T cells to enhance intense tumoricidal activity and persistence. CD19.28.40z CAR-T cells promoted pNF-κB and pRelB downstream signaling while diminishing NFAT signaling upon antigen exposure. CD19.28.40z CAR-T cells demonstrated greater proliferation, which translated into effective anti-tumor cytotoxicity in long-term co-culture assay. Repetitive weekly antigen stimulation unveiled continuous CAR-T cell expansion while preserving central memory T cell subset and lower expression of exhaustion phenotypes. The intrinsic genes underlying CD19.28.40z CAR-T cell responses were compared with conventional CARs and demonstrated the up-regulated genes associated with T cell proliferation and memory as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway. Enrichment of genes toward T cell stemness, particularly SELL, IL-7r, TCF7, and KLF2, was observed. Effective and continuing anti-tumor cytotoxicity in vivo was exhibited in both B cell lymphoblastic leukemia and B cell non-Hodgkin lymphoma xenograft models while demonstrating persistent T cell memory signatures. The functional enhancement of CD37.28.40z CAR-T cell activities against CD37+ tumor cells was further validated. The modification of dual T/B cell signaling molecules remarkably maximized the efficacy of CAR-T cell therapy.

Mtb HLA-E-tetramer-sorted CD8+ T cells have a diverse TCR repertoire.

HLA-E molecules can present self- and pathogen-derived peptides to both natural killer (NK) cells and T cells. T cells that recognize HLA-E peptides via their T cell receptor (TCR) are termed donor-unrestricted T cells due to restricted allelic variation of HLA-E. The composition and repertoire of HLA-E TCRs is not known so far. We performed TCR sequencing on CD8+ T cells from 21 individuals recognizing HLA-E tetramers (TMs) folded with two Mtb-HLA-E-restricted peptides. We sorted HLA-E Mtb TM+ and TM- CD8+ T cells directly ex vivo and performed bulk RNA-sequencing and single-cell TCR sequencing. The identified TCR repertoire was diverse and showed no conservation between and within individuals. TCRs selected from our single-cell TCR sequencing data could be activated upon HLA-E/peptide stimulation, although not robust, reflecting potentially weak interactions between HLA-E peptide complexes and TCRs. Thus, HLA-E-Mtb-specific T cells have a highly diverse TCR repertoire.

Transcriptional reprogramming via signaling domains of CD2, CD28, and 4-1BB.

Costimulatory signals provided to T cells during antigen encounter have a decisive role in the outcome of immune responses. Here, we used chimeric receptors harboring the extracellular domain of mouse inducible T cell costimulator (mICOS) to study transcriptional activation mediated by cytoplasmic sequences of the major T cell costimulatory receptors CD28, 4-1BB, and CD2. The chimeric receptors were introduced in a T cell reporter platform that allows to simultaneously evaluate nuclear factor κB (NF-κB), NFAT, and AP-1 activation. Engagement of the chimeric receptors induced distinct transcriptional profiles. CD28 signaling activated all three transcription factors, whereas 4-1BB strongly promoted NF-κB and AP-1 but downregulated NFAT activity. CD2 signals resulted in the strongest upregulation of NFAT. Transcriptome analysis revealed pronounced and distinct gene expression signatures upon CD2 and 4-1BB signaling. Using the intracellular sequence of CD28, we exemplify that distinct signaling motifs endow chimeric receptors with different costimulatory capacities.

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.

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.

A high-throughput two-cell assay for interrogating inhibitory signaling pathways in T cells.

The recent success of immunotherapies relying on manipulation of T-cell activation highlights the value of characterising the mediators of immune checkpoint signaling. CRISPR/Cas9 is a popular approach for interrogating signaling pathways; however, the lack of appropriate assays for studying inhibitory signaling in T cells is limiting the use of large-scale perturbation-based approaches. Here, we adapted an existing Jurkat cell-based transcriptional reporter assay to study both activatory and inhibitory (PD-1-mediated) T-cell signaling using CRISPR-based genome screening in arrayed and pooled formats. We targeted 64 SH2 domain-containing proteins expressed by Jurkat T cells in an arrayed screen, in which individual targets could be assessed independently, showing that arrays can be used to study mediators of both activatory and inhibitory signaling. Pooled screens succeeded in simultaneously identifying many of the known mediators of proximal activating and inhibitory T-cell signaling, including SHP2 and PD-1, confirming the utility of the method. Altogether, the data suggested that SHP2 is the major PD-1-specific, SH2 family mediator of inhibitory signaling. These approaches should allow the systematic analysis of signaling pathways in T cells.

Interrogating ligand-receptor interactions using highly sensitive cellular biosensors.

Interactions of membrane-resident proteins are important targets for therapeutic interventions but most methods to study them are either costly, laborious or fail to reflect the physiologic interaction of membrane resident proteins in trans. Here we describe highly sensitive cellular biosensors as a tool to study receptor-ligand pairs. They consist of fluorescent reporter cells that express chimeric receptors harboring ectodomains of cell surface molecules and intracellular signaling domains. We show that a broad range of molecules can be integrated into this platform and we demonstrate its applicability to highly relevant research areas, including the characterization of immune checkpoints and the probing of cells for the presence of receptors or ligands. The platform is suitable to evaluate the interactions of viral proteins with host receptors and to test for neutralization capability of drugs or biological samples. Our results indicate that cellular biosensors have broad utility as a tool to study protein-interactions.

The ligand-dependent suppression of TCR signaling by the immune checkpoint receptor LAG3 depends on the cytoplasmic RRFSALE motif.

Lymphocyte activation gene 3 (LAG3) is an inhibitory immune checkpoint receptor that restrains autoimmune and antitumor responses, but its evolutionarily conserved cytoplasmic tail lacks classical inhibitory motifs. Major histocompatibility complex class II (MHC class II) is an established LAG3 ligand, and fibrinogen-like protein 1 (FGL1), lymph node sinusoidal endothelial cell C-type lectin (LSECtin), and Galectin-3 have been proposed as alternative binding partners that play important roles in LAG3 function. Here, we used a fluorescent human T cell reporter system to study the function of LAG3. We found that LAG3 reduced the response to T cell receptor stimulation in the presence of MHC class II molecules to a lesser extent compared with the receptor programmed cell death protein 1. Analysis of deletion mutants demonstrated that the RRFSALE motif in the cytoplasmic tail of LAG3 was necessary and sufficient for LAG3-mediated inhibition. In this system, FGL1, but not LSECtin or Galectin-3, acted as a LAG3 ligand that weakly induced inhibition. LAG3-blocking antibodies attenuated LAG3-mediated inhibition in our reporter cells and enhanced reporter cell activation even in the absence of LAG3 ligands, indicating that they could potentially enhance T cell responses independently of their blocking effect.

FcγR requirements and costimulatory capacity of Urelumab, Utomilumab, and Varlilumab.

Introduction: Targeting costimulatory receptors of the tumor necrosis factor receptor (TNFR) superfamily with agonistic antibodies is a promising approach in cancer immuno therapy. It is known that their efficacy strongly depends on FcγR cross-linking. Methods: In this study, we made use of a Jurkat-based reporter platform to analyze the influence of individual FcγRs on the costimulatory activity of the 41BB agonists, Urelumab and Utomilumab, and the CD27 agonist, Varlilumab. Results: We found that Urelumab (IgG4) can activate 41BB-NFκB signaling without FcγR cross-linking, but the presence of the FcγRs (CD32A, CD32B, CD64) augments the agonistic activity of Urelumab. The human IgG2 antibody Utomilumab exerts agonistic function only when crosslinked via CD32A and CD32B. The human IgG1 antibody Varlilumab showed strong agonistic activity with all FcγRs tested. In addition, we analyzed the costimulatory effects of Urelumab, Utomilumab, and Varlilumab in primary human peripheral blood mononuclear cells (PBMCs). Interestingly, we observed a very weak capacity of Varlilumab to enhance cytokine production and proliferation of CD4 and CD8 T cells. In the presence of Varlilumab the percentage of annexin V positive T cells was increased, indicating that this antibody mediated FcγR-dependent cytotoxic effects. Conclusion: Collectively, our data underscore the importance to perform studies in reductionist systems as well as in primary PBMC samples to get a comprehensive understanding of the activity of costimulation agonists.

Human intestine and placenta exhibit tissue-specific expression of RAGE isoforms.

The receptor for advanced glycation end products (RAGE) is encoded by AGER, a gene that is subjected to tissue-specific alternative splicing. Splice variants of RAGE in intestine and placenta are unknown and contradictory data concerning RAGE protein expression in these tissues have been published. As a basis for future functional studies, we examined RAGE expression in small intestine, colon and placentas. PCR cloning revealed that full-length RAGE is the only RAGE transcript isoform expressed in placenta. In the small intestine, the major transcript isoform detected was RAGE_v1 encoding the C-terminally truncated soluble receptor. In the colon, both full-length RAGE as well as several splice variants were identified. Four antibodies were used to study protein expression by immunoblotting and were carefully validated. Appropriate controls were essential to avoid misinterpretation of bands caused by non-specific reactivity of antibodies. Only one of four antibodies tested detected full-length RAGE in placenta, whereas no RAGE-specific band was detected in intestinal tissues despite loading >30-fold more intestinal tissue than the positive control, human lung. RAGE expression levels in the placenta were 100-fold lower compared with human lung when analyzed by ELISA, and no significant differences in RAGE expression were detected between healthy placentas and placentas from women with preeclampsia, gestational diabetes mellitus, or fetal growth restriction. We conclude that healthy placental chorionic tissue expresses low levels of full-length RAGE, whereas expression of the tissue-specific intestinal isoforms is below the limit of detection. Low RAGE expression levels in combination with a lack of antibody validation may explain the conflicting published results on RAGE protein expression in intestine and placenta.

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.

Using the Jurkat reporter T cell line for evaluating the functionality of novel chimeric antigen receptors.

Background: T cells that are genetically modified with chimeric antigen receptor (CAR) hold promise for immunotherapy of cancer. Currently, there are intense efforts to improve the safety and efficacy of CAR T cell therapies against liquid and solid tumors. Earlier we designed a novel CAR backbone (FiCAR) where the spacer is derived from immunoglobulin (Ig) -like domains of the signal-regulatory protein alpha (SIRPα). However, the analysis of novel CAR using primary T cells is slow and laborious. Methods: To explore the versatility of the CAR backbone, we designed a set of variant FiCARs with different spacer lengths and targeting antigens. To expedite the analysis of the novel CARs, we transduced the FiCAR genes using lentiviruses into Jurkat reporter T cells carrying fluorescent reporter genes. The expression of fluorescent markers in response to FiCAR engagement with targets was analyzed by flow cytometry, and cytotoxicity was evaluated using killing assays. Furthermore, the killing mechanisms that are employed by FiCAR-equipped Jurkat T cells were investigated by flow cytometry, and the intracellular pathways involved in signaling by FiCAR were analyzed by phosphoproteomic analysis using mass spectrometry. Results: Seven different CARs were designed and transduced into Jurkat reporter cells. We show that the SIRPα derived FiCARs can be detected by flow cytometry using the SE12B6A4 antibody recognizing SIRPα. Furthermore, FiCAR engagement leads to robust activation of NFκß and NFAT signaling, as demonstrated by the expression of the fluorescent reporter genes. Interestingly, the Jurkat reporter system also revealed tonic signaling by a HER-2 targeting FiCAR. FiCAR-equipped Jurkat T cells were cytotoxic in cocultures with target cells and target cell engagement lead to an upregulation of CD107a on the Jurkat reporter T cell surface. Phosphoproteomic analyses confirmed signal transduction via the intracellular CD28/CD3ζ sequences upon the interaction of the FiCAR1 with its antigen. In addition, downstream signaling of CD3ζ/ZAP70- SLP-76-PLCγ, PI3K-AKT-NFκB pathways and activation of NFAT and AP-1 were observed. Conclusion: We conclude that the FiCAR backbone can be shortened and lengthened at will by engineering it with one to three SIRPα derived Ig-like domains, and the FiCARs are functional when equipped with different single chain variable fragment target binding domains. The Jurkat reporter system expedites the analysis of novel CARs as to their expression, signaling function, evaluation of tonic signaling issues and cytotoxic activity.

Interoperability of RTN1A in dendrite dynamics and immune functions in human Langerhans cells.

Skin is an active immune organ where professional antigen-presenting cells such as epidermal Langerhans cells (LCs) link innate and adaptive immune responses. While Reticulon 1A (RTN1A) was recently identified in LCs and dendritic cells in cutaneous and lymphoid tissues of humans and mice, its function is still unclear. Here, we studied the involvement of this protein in cytoskeletal remodeling and immune responses toward pathogens by stimulation of Toll-like receptors (TLRs) in resident LCs (rLCs) and emigrated LCs (eLCs) in human epidermis ex vivo and in a transgenic THP-1 RTN1A+ cell line. Hampering RTN1A functionality through an inhibitory antibody induced significant dendrite retraction of rLCs and inhibited their emigration. Similarly, expression of RTN1A in THP-1 cells significantly altered their morphology, enhanced aggregation potential, and inhibited the Ca2+ flux. Differentiated THP-1 RTN1A+ macrophages exhibited long cell protrusions and a larger cell body size in comparison to wild-type cells. Further, stimulation of epidermal sheets with bacterial lipoproteins (TLR1/2 and TLR2 agonists) and single-stranded RNA (TLR7 agonist) resulted in the formation of substantial clusters of rLCs and a significant decrease of RTN1A expression in eLCs. Together, our data indicate involvement of RTN1A in dendrite dynamics and structural plasticity of primary LCs. Moreover, we discovered a relation between activation of TLRs, clustering of LCs, and downregulation of RTN1A within the epidermis, thus indicating an important role of RTN1A in LC residency and maintaining tissue homeostasis.

BTLA inhibition has a dominant role in the cis-complex of BTLA and HVEM.

The engagement of the herpesvirus entry mediator (HVEM, TNFRSF14) by the B and T lymphocyte attenuator (BTLA) represents a unique interaction between an activating receptor of the TNFR-superfamily and an inhibitory receptor of the Ig-superfamily. BTLA and HVEM have both been implicated in the regulation of human T cell responses, but their role is complex and incompletely understood. Here, we have used T cell reporter systems to dissect the complex interplay of HVEM with BTLA and its additional ligands LIGHT and CD160. Co-expression with LIGHT or CD160, but not with BTLA, induced strong constitutive signaling via HVEM. In line with earlier reports, we observed that in cis interaction of BTLA and HVEM prevented HVEM co-stimulation by ligands on surrounding cells. Intriguingly, our data indicate that BTLA mediated inhibition is not impaired in this heterodimeric complex, suggesting a dominant role of BTLA co-inhibition. Stimulation of primary human T cells in presence of HVEM ligands indicated a weak costimulatory capacity of HVEM potentially owed to its in cis engagement by BTLA. Furthermore, experiments with T cell reporter cells and primary T cells demonstrate that HVEM antibodies can augment T cell responses by concomitantly acting as checkpoint inhibitors and co-stimulation agonists.

Discovery of the first PD-1 ligand encoded by a pathogen.

Large double-stranded DNA viruses deploy multiple strategies to subvert host immune defenses. Some of these tactics are mediated by viral gene products acquired by horizontal gene transfer from the corresponding hosts and shaped throughout evolution. The programmed death-1 (PD-1) receptor and its ligands, PD-L1 and PD-L2, play a pivotal role attenuating T-cell responses and regulating immune tolerance. In this study, we report the first functional PD-L1 homolog gene (De2) found in a pathogen. De2, captured by a γ-herpesvirus from its host during co-evolution around 50 million years ago, encodes a cell-surface glycoprotein that interacts with high affinity and stability with host PD-1. We also find that mutations evolved by the viral protein result in a significant loss of its ability to interact in cis with CD80, an interaction that for PD-L1:CD80 has been reported to block PD-1 inhibitory pathways. Furthermore, we demonstrate that the viral protein strongly inhibits T-cell signaling. Our observations suggest that PD-L1 homologs may enable viruses to evade T cell responses, favor their replication, and prevent excessive tissue damage. Altogether, our findings reveal a novel viral immunosuppressive strategy and highlight the importance of the modulation of the PD-1/PD-L1 axis during viral infections.

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.

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.