Strong Effector Functions Research Articles

Generation of non-genetically modified, CAR-like, NK cells

BackgroundNatural killer (NK) cell therapy is considered an attractive and safe strategy for anticancer therapy. Nevertheless, when autologous or allogenic NK cells are used alone, the clinical benefit has been...

A viral vaccine design harnessing prior BCG immunization confers protection against Ebola virus.

Previous studies have demonstrated the efficacy and feasibility of an anti-viral vaccine strategy that takes advantage of pre-existing CD4+ helper T (Th) cells induced by Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination. This strategy uses immunization with recombinant fusion proteins comprised of a cell surface expressed viral antigen, such as a viral envelope glycoprotein, engineered to contain well-defined BCG Th cell epitopes, thus rapidly recruiting Th cells induced by prior BCG vaccination to provide intrastructural help to virus-specific B cells. In the current study, we show that Th cells induced by BCG were localized predominantly outside of germinal centers and promoted antibody class switching to isotypes characterized by strong Fc receptor interactions and effector functions. Furthermore, BCG vaccination also upregulated FcγR expression to potentially maximize antibody-dependent effector activities. Using a mouse model of Ebola virus (EBOV) infection, this vaccine strategy provided sustained antibody levels with strong IgG2c bias and protection against lethal challenge. This general approach can be easily adapted to other viruses, and may be a rapid and effective method of immunization against emerging pandemics in populations that routinely receive BCG vaccination.

A viral vaccine design harnessing prior BCG immunization confers protection against Ebola virus.

Previous studies have demonstrated the efficacy and feasibility of an anti-viral vaccine strategy that takes advantage of pre-existing CD4 + helper T (Th) cells induced by Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccination. This strategy uses immunization with recombinant fusion proteins comprised of a cell surface expressed viral antigen, such as a viral envelope glycoprotein, engineered to contain well-defined BCG Th cell epitopes, thus rapidly recruiting Th cells induced by prior BCG vaccination to provide intrastructural help to virus-specific B cells. In the current study, we show that Th cells induced by BCG were localized predominantly outside of germinal centers and promoted antibody class switching to isotypes characterized by strong Fc receptor interactions and effector functions. Furthermore, BCG vaccination also upregulated FcγR expression to potentially maximize antibody-dependent effector activities. Using a mouse model of Ebola virus (EBOV) infection, this vaccine strategy provided sustained antibody levels with strong IgG2c bias and protection against lethal challenge. This general approach can be easily adapted to other viruses, and may be a rapid and effective method of immunization against emerging pandemics in populations that routinely receive BCG vaccination.

Long-term humoral signatures following acute pediatric COVID-19 and Multisystem Inflammatory Syndrome in Children.

Although most children experience mild symptoms during acute SARS-CoV-2 infection, some develop the severe post-COVID-19 complication, Multisystem Inflammatory Syndrome in Children (MIS-C). While acute presentations of COVID-19 and MIS-C have been well immunophenotyped, little is known about the lasting immune profile in children after acute illness. Children 2 months-20 years of age presenting with either acute COVID-19 (n = 9) or MIS-C (n = 12) were enrolled in a Pediatric COVID-19 Biorepository at a single medical center. We deeply profiled humoral immune responses and circulating cytokines following pediatric COVID-19 and MIS-C. Twenty-one children and young adults provided blood samples at both acute presentation and 6-month follow-up (mean: 6.5 months; standard deviation: 1.77 months). Pro-inflammatory cytokine elevations resolved after both acute COVID-19 and MIS-C. Humoral profiles continue to mature after acute COVID-19, displaying decreasing IgM and increasing IgG over time, as well as stronger effector functions, including antibody-dependent monocyte activation. In contrast, MIS-C immune signatures, especially anti-Spike IgG1, diminished over time. Here, we show the mature immune signature after pediatric COVID-19 and MIS-C, displaying resolving inflammation with recalibration of the humoral responses. These humoral profiles highlight immune activation and vulnerabilities over time in these pediatric post-infectious cohorts. The pediatric immune profile matures after both COVID-19 and MIS-C, suggesting a diversified anti-SARS-CoV-2 antibody response after resolution of acute illness. While pro-inflammatory cytokine responses resolve in the months following acute infection in both conditions, antibody-activated responses remain relatively heightened in convalescent COVID-19. These data may inform long-term immunoprotection from reinfection in children with past SARS-CoV-2 infections or MIS-C.

Regulation of effector and memory CD8 + T cell differentiation: a focus on orphan nuclear receptor NR4A family, transcription factor, and metabolism.

CD8 + T cells undergo rapid expansion followed by contraction and the development of memory cells after their receptors are activated. The development of immunological memory following acute infection is a complex phenomenon that involves several molecular, transcriptional, and metabolic mechanisms. As memory cells confer long-term protection and respond to secondary stimulation with strong effector function, understanding the mechanisms that influence their development is of great importance. Orphan nuclear receptors, NR4As, are immediate early genes that function as transcription factors and bind with the NBRE region of chromatin. Interestingly, the NBRE region of activated CD8 + T cells is highly accessible at the same time the expression of NR4As is induced. This suggests a potential role of NR4As in the early events post T cell activation that determines cell fate decisions. In this review, we will discuss the influence of NR4As on the differentiation of CD8 + T cells during the immune response to acute infection and the development of immunological memory. We will also discuss the signals, transcription factors, and metabolic mechanisms that control cell fate decisions. HIGHLIGHTS: Memory CD8 + T cells are an essential subset that mediates long-term protection after pathogen encounters. Some specific environmental cues, transcriptional factors, and metabolic pathways regulate the differentiation of CD8 + T cells and the development of memory cells. Orphan nuclear receptor NR4As are early genes that act as transcription factors and are highly expressed post-T cell receptor activation. NR4As influence the effector function and differentiation of CD8 + T cells and also control the development of immunological memory following acute infection.

The adaptive immune system in early life: The shift makes it count.

Respiratory infectious diseases encountered early in life may result in life-threatening disease in neonates, which is primarily explained by the relatively naive neonatal immune system. Whereas vaccines are not readily available for all infectious diseases, vaccinations have greatly reduced childhood mortality. However, repeated vaccinations are required to reach protective immunity in infants and not all vaccinations are effective at young age. Moreover, protective adaptive immunity elicited by vaccination wanes more rapidly at young age compared to adulthood. The infant adaptive immune system has previously been considered immature but this paradigm has changed during the past years. Recent evidence shows that the early life adaptive immune system is equipped with a strong innate-like effector function to eliminate acute pathogenic threats. These strong innate-like effector capacities are in turn kept in check by a tolerogenic counterpart of the adaptive system that may have evolved to maintain balance and to reduce collateral damage. In this review, we provide insight into these aspects of the early life's adaptive immune system by addressing recent literature. Moreover, we speculate that this shift from innate-like and tolerogenic adaptive immune features towards formation of immune memory may underlie different efficacy of infant vaccination in these different phases of immune development. Therefore, presence of innate-like and tolerogenic features of the adaptive immune system may be used as a biomarker to improve vaccination strategies against respiratory and other infections in early life.

A Microfluidic Approach for Probing Heterogeneity in Cytotoxic T-Cells by Cell Pairing in Hydrogel Droplets.

Cytotoxic T-cells (CTLs) exhibit strong effector functions to leverage antigen-specific anti-tumoral and anti-viral immunity. When naïve CTLs are activated by antigen-presenting cells (APCs) they display various levels of functional heterogeneity. To investigate this, we developed a single-cell droplet microfluidics platform that allows for deciphering single CTL activation profiles by multi-parameter analysis. We identified and correlated functional heterogeneity based on secretion profiles of IFNγ, TNFα, IL-2, and CD69 and CD25 surface marker expression levels. Furthermore, we strengthened our approach by incorporating low-melting agarose to encapsulate pairs of single CTLs and artificial APCs in hydrogel droplets, thereby preserving spatial information over cell pairs. This approach provides a robust tool for high-throughput and single-cell analysis of CTLs compatible with flow cytometry for subsequent analysis and sorting. The ability to score CTL quality, combined with various potential downstream analyses, could pave the way for the selection of potent CTLs for cell-based therapeutic strategies.

Expansion and Enrichment of Gamma-Delta (γδ) T Cells from Apheresed Human Product

Although Vγ9Vδ2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (γδ) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure γδ T cells drug product for first-in-human use of allogeneic γδ T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. The lymphocyte-rich fraction is utilized, and the γδ T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of γδ T cells, a clinical-grade magnetic cell-separation device and TCRαβ beads are used to deplete contaminating T-cell receptor (TCR)αβ T cells. The highly enriched γδ T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched γδ T cells in co-culture with the aAPCs facilitates the manufacture of highly pure γδ T cells with an average fold expansion of >229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta (γδ) T Cells from Apheresed Human Product.

Although Vγ9Vδ2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (γδ) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure γδ T cells drug product for first-in-human use of allogeneic γδ T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. The lymphocyte-rich fraction is utilized, and the γδ T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of γδ T cells, a clinical-grade magnetic cell-separation device and TCRαβ beads are used to deplete contaminating T-cell receptor (TCR)αβ T cells. The highly enriched γδ T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched γδ T cells in co-culture with the aAPCs facilitates the manufacture of highly pure γδ T cells with an average fold expansion of >229,000-fold from healthy donor blood.

The TGF-β/SMAD Signaling Pathway As a Mediator of NK Cell Dysfunction and Immune Evasion in Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a spectrum of clonal hematopoietic disorders affecting the myeloid lineage, characterized by ineffective hematopoiesis and dysplastic features. In about a third of patients, MDS transforms to secondary AML, and for this subgroup the overall survival is dismal. Immune responses against MDS have the potential to prevent disease progression; however, immune evasion mechanisms pose a formidable barrier. In fact, NK cells from MDS patients show significant impairment in their effector function but the mechanism underlying this immune dysfunction is not well understood. Although a multiplicity of mechanisms contribute to NK cell immune evasion, two of the most important mechanisms are down-regulation of activating receptors and the production of inhibitory cytokines such as transforming growth factor β1 (TGF-β), a broadly immunosuppressive cytokine that renders NK cells unable to kill tumor cells. We hypothesized that the MDS microenvironment evades NK cell immune surveillance through a mechanism involving TGF-β, and that this immune dysfunction can be reversed by agents that interfere with this inhibitory pathway.Thus, we isolated NK cells from the peripheral blood of MDS patients (n=16) and compared them to the NK cells from healthy donors (n=10). We observed that NK cells from high-risk MDS patients are dysfunctional, produce significantly less cytokines IFNg (p<0.0001) and CD107a (p=0.0029) and display significantly lower cytotoxicity (p=0.04) towards K562 as demonstrated by 51-Cr release assay compared to their healthy counterparts. This was associated with an abnormal phenotype with downregulation of many activating receptors and upregulation of exhaustion/inhibitory molecules such as PD-1 and the TGF-β induced molecules, CD9 and CD103. To show direct involvement of the TGF-β pathway in mediating MDS-induced NK dysfunction , w e examined activation of the TGF-β pathway by measuring phosphorylation of SMAD 2/3and showed that phospho-SMAD 2/3 is constitutively expressed in NK cells from MDS patients but not in healthy donor NK cells (p=0.03). We noted high expression of TGF-β LAP on the surface of myeloid blasts cells from MDS patients (80-90%) but not healthy myeloid cells (p <0.0001). Interestingly, when myeloid blasts were cultured together with NK cells, we detected significantly larger amounts of TGF-β in the supernatant, suggesting that NK-blast cross-talk may modulate TGF-β release by myeloid blasts. To further explore the mechanism of TGF-β-induced NK dysfunction and the contribution of NK-blast cross-talk to this phenomenon, we co-cultured healthy donor NK cells with myeloid blasts either in direct contact or separated using a transwell membrane and measured their cytotoxicity and cytokine release after 72h of co-culture. NK cells cultured in direct contact with myeloid blasts had significantly impaired effector function as evidenced by lower cytokine production (TNFa p<0.0001, IFNg p=0.0003) and CD107a degranulation (p=0.0002), whereas NK cells separated from blasts by a transwell membrane did not show any evidence of dysfunction. These data point to an important role for cell-cell contact in mediating blast-induced NK dysfunction.We next investigated whether targeted inhibition of TGF-β can protect NK cells from TGF-β-mediated dysfunction, using two different approaches. First, we tested the role of a TGFβR1 kinase inhibitor, Galunisertib, currently in clinical trials for MDS. The addition of Galunisertib to cocultures of healthy NK cells and myeloid blasts prevented NK cell dysfunction and blast-induced SMAD2/3 phosphorylation. For the second approach, we tested if TGF-βR2 knockout by CRISPR-CAS9 gene editing can render them resistant to the suppressive effect of TGF-β. When co-cultured with 50ng/ml of recombinant TGF-β or myeloid blasts for 48 hrs, TGF-βR2 KO NK cells retained their ability to mediate a strong effector function. In vivo experiments to assess the in vivo activity of TGF-βR2 KO NK cells in a xenograft model of myeloid leukemia are underway.Together, these data support an important role for the TGF-β/SMAD signaling pathway as a mediator of NK cell dysfunction and hence immune evasion in MDS, and support strategies to target the TGF-β/SMAD signaling axis for next-generation NK cell therapies in MDS. DisclosuresKantarjian:Bristol-Meyers Squibb: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; ARIAD: Research Funding; Delta-Fly Pharma: Research Funding; Amgen: Research Funding.

Biolayer Interferometry-based FcγRIIa binding assay for a therapeutic antibody with strong effector function

FcγRIIa receptor binding is part of the mechanism of action for many therapeutic antibodies. AlphaScreen® technology and Biolayer Interferometry (BLI) are often used to assess protein-protein interactions. Recently we demonstrated that the presence of aggregates in samples significantly increased binding potency values in AlphaScreen®-based FcRn binding assays, sometimes masking the loss of potency. Even bigger effect of aggregates was observed in an AlphaScreen®-based FcγRIIa binding assay for a monoclonal antibody with strong effector function. To resolve this issue a novel BLI-based FcγRIIa binding assay was developed and qualified. The assay measures association binding responses and calculates the binding potency of the samples relative to the standard using Parallel Line Analysis. The method overcomes interference of aggregates present in the samples, distinguishes different Fc glycosylation patterns, and is stability-indicating. It can be used for sample characterization, drug product release and stability testing.

Effector function of anti-pyroglutamate-3 A\u03b2 antibodies affects cognitive benefit, glial activation and amyloid clearance in Alzheimer\u2019s-like mice

BackgroundPyroglutamate-3 Aβ (pGlu-3 Aβ) is an N-terminally truncated and post-translationally modified Aβ species found in Alzheimer’s disease (AD) brain. Its increased peptide aggregation propensity and toxicity make it an attractive emerging treatment strategy for AD. We address the question of how the effector function of an anti-pGlu-3 Aβ antibody influences the efficacy of immunotherapy in mouse models with AD-like pathology.MethodsWe compared two different immunoglobulin (Ig) isotypes of the same murine anti-pGlu-3 Aβ mAb (07/1 IgG1 and 07/2a IgG2a) and a general N-terminal Aβ mAb (3A1 IgG1) for their ability to clear Aβ and protect cognition in a therapeutic passive immunotherapy study in aged, plaque-rich APPSWE/PS1ΔE9 transgenic (Tg) mice. We also compared the ability of these antibodies and a CDC-mutant form of 07/2a (07/2a-k), engineered to avoid complement activation, to clear Aβ in an ex vivo phagocytosis assay and following treatment in APPSLxhQC double Tg mice, and to activate microglia using longitudinal microPET imaging with TSPO-specific 18F-GE180 tracer following a single bolus antibody injection in young and old Tg mice.ResultsWe demonstrated significant cognitive improvement, better plaque clearance, and more plaque-associated microglia in the absence of microhemorrhage in aged APPSWE/PS1ΔE9 Tg mice treated with 07/2a, but not 07/1 or 3A1, compared to PBS in our first in vivo study. All mAbs cleared plaques in an ex vivo assay, although 07/2a promoted the highest phagocytic activity. Compared with 07/2a, 07/2a-k showed slightly reduced affinity to Fcγ receptors CD32 and CD64, although the two antibodies had similar binding affinities to pGlu-3 Aβ. Treatment of APPSLxhQC mice with 07/2a and 07/2a-k mAbs in our second in vivo study showed significant plaque-lowering with both mAbs. Longitudinal 18F-GE180 microPET imaging revealed different temporal patterns of microglial activation for 3A1, 07/1, and 07/2a mAbs and no difference between 07/2a-k and PBS-treated Tg mice.ConclusionOur results suggest that attenuation of behavioral deficits and clearance of amyloid is associated with strong effector function of the anti-pGlu-3 Aβ mAb in a therapeutic treatment paradigm. We present evidence that antibody engineering to reduce CDC-mediated complement binding facilitates phagocytosis of plaques without inducing neuroinflammation in vivo. Hence, the results provide implications for tailoring effector function of humanized antibodies for clinical development.

Phenotypic and Functional Analysis of Human NK Cell Subpopulations According to the Expression of FcεRIγ and NKG2C.

Human memory-like NK cells are commonly defined by either a lack of FcεRIγ or gain of NKG2C expression. Here, we investigated the heterogeneity of human CD56dim NK cell subpopulations according to the expression of FcεRIγ and NKG2C in a large cohort (n = 127). Although the frequency of FcεRIγ− and NKG2C+ NK cells positively correlated, the FcεRIγ− and NKG2C+ NK cell populations did not exactly overlap. The FcεRIγ+NKG2C+, FcεRIγ−NKG2C+, and FcεRIγ−NKG2C− NK cell populations were only evident after HCMV infection, but each had distinct characteristics. Among the subpopulations, FcεRIγ−NKG2C+ NK cells exhibited the most restricted killer immunoglobulin-like receptor repertoire, suggesting clonal expansion. Moreover, FcεRIγ−NKG2C+ NK cells exhibited the lowest Ki-67 and highest Bcl-2 expression, indicating the long-lived quiescent memory-like property. Functionally, FcεRIγ−NKG2C+ NK cells had weak natural effector function against K562 but strong effector functions by CD16 engagement, whereas FcεRIγ+NKG2C+ NK cells had strong effector functions in both settings. Anatomically, the FcεRIγ+NKG2C+, FcεRIγ−NKG2C+, and FcεRIγ−NKG2C− NK cell populations were present in multiple human peripheral organs. In conclusion, we demonstrate the heterogeneity of memory-like NK cells stratified by FcεRIγ and NKG2C and suggest both markers be utilized to better define these cells.

Priming of HIV-1-specific CD8+ T cells with strong functional properties from naïve T cells.

BackgroundHIV-1-specific CD8+ T cells are required for immune suppression of HIV-1 replication and elimination of the associated viral reservoirs. However, effective induction of functional HIV-1-specific CD8+ T cells from naïve cells remains problematic in the setting of human vaccine trials. In this study, we investigated priming of functional HIV-1-specific CD8+ T cells from naïve cells.MethodsHIV-1-specific CD8+ T cells were primed from naïve T cells of HIV-1-seronegative individuals using TLR4 ligand LPS or STING ligand 3′3′-cGAMP in vitro. We established HIV-1-specific CD8+ T cell lines from primed T cells and then investigated functional properties of these cells.FindingsHIV-1-specific CD8+ T cells primed with LPS failed to suppress HIV-1. In contrast, 3′3′-cGAMP effectively primed HIV-1-specific CD8+ T cells with strong ability to suppress HIV-1. 3′3′-cGAMP-primed T cells had higher expression levels of perforin and granzyme B than LPS-primed ones. The expression levels of granzyme B and perforin and viral suppression ability of 3′3′-cGAMP-primed T cells were positively correlated with the production level of type I IFN from PBMCs stimulated with 3′3′-cGAMP.InterpretationThe present study demonstrates the potential of 3′3′-cGAMP to induce HIV-1-specific CD8+ T cells with strong effector function from naïve cells via a strong type I IFN production and suggests that this STING ligand may be useful for AIDS vaccine and cure treatment.

Calibrated CAR Activation Potential Directs Alternative T Cell Fates and Therapeutic Potency

Immunotherapy with second-generation chimeric antigen receptor (CAR) T cells has achieved great clinical success against hematological malignancies. CD19-specific CARs incorporating CD28 and CD3z signaling domains have demonstrated remarkable potency resulting in the frequent induction of complete remissions and were recently approved by the US FDA for use in some refractory B cell malignancies. We previously demonstrated that CD28-based CARs program strong effector functions, but impart a relatively limited T cell lifespan. Increasing functional T cell persistence is therefore likely to further enhance the therapeutic success of 1928z CAR T cells.We hypothesized that excessive signal strength arising from redundancy of combined CD3z and CD28 signals might foster terminal T cell differentiation and exhaustion. We therefore proceeded to titrate the activation potential of CD28-based CARs and assess the impact thereof on the function, longevity and therapeutic potency of CAR T cells.We analyzed the contribution of individual immunoreceptor tyrosine-based activation motifs (ITAMs) to the phenotype and function of 1928z CAR T cells. ITAM-mutated CAR T cells demonstrated similar expression levels in retrovirally transduced primary T cells and directed comparable short-term cytotoxicity and proliferation capacity in vitro. However, remarkable differences in their therapeutic potency emerged when T cells expressing different mutant CARs were tested in the pre-B acute lymphoblastic leukemia NALM6 mouse model. The CAR “stress test” model revealed that a CAR containing a single functional ITAM - depending on its position - outperformed wild-type 1928z CARs, achieving rapid and durable tumor eradication even at low T cell doses, by delaying T cell differentiation and exhaustion. CAR T cells retrieved from the bone marrow of treated mice demonstrated that inactivation of two ITAM domains augmented CAR persistence at the tumor sites with a higher percentage of central memory cells and a decreased proportion of terminally differentiated effector cells. Deletion mutants further revealed the importance of ITAM location within second-generation CAR T cells.These findings were rigorously tested by directing the mutant CAR cDNAs to the T-cell receptor α constant (TRAC) locus using CRISPR/Cas9 technology, thereby ruling out potentially confounding effects arising from different CAR expression levels. TRAC-1928z mutants demonstrated superior antitumor efficacy compared to conventional TRAC-1928z CARs and prevented terminal T cell differentiation and exhaustion.Genome-wide transcriptional profiles of TRAC-edited naïve peripheral blood T cells further demonstrated that CARs encoding different ITAMs direct T cells to different fates. While TRAC-1928z CARs demonstrated similarity to transcriptional profiles of effector cells, reducing the number of ITAMs to one ITAM preserved a less-differentiated T cell state and promoted greater T cell persistence. We identified one 1928z mutant CAR, which improved therapeutic potency and induced a transcriptional profile similar to that of stem cell memory T cells (TSCM). Another 1928z mutant CAR with further reduction of the activation potential resulted in a naïve-like phenotype with great proliferation potential and persistence, but greatly diminished anti-tumor efficacy.In conclusion, we demonstrate that the number and position of ITAMs in 1928z CAR T cells influence functional, phenotypic and transcriptional programs resulting in profound effects on therapeutic potency. Balancing T cell differentiation and acquisition of effector functions is essential to optimize therapeutic potency of CAR T cells and can be intrinsically regulated by defined mutations in the CD3z chain of 1928z CAR T cells.Improved therapeutic potency of CAR T cells can thus be achieved by calibrating activation strength, thus retaining memory functions and preventing exhaustion, without compromising effector functions. Importantly, we were able to identify a novel CAR design which programs a favorable balance of effector and memory signatures, inducing increased persistence of highly functional CARs with the replicative capacity of long-lived memory cells and potent effector functions. Clinical studies evaluating the new CAR design are in preparation. DisclosuresSadelain:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Fate Therapeutics Inc.: Research Funding.

CD137 (4-1BB) Signalosome: Complexity Is a Matter of TRAFs.

CD137 (4-1BB, Tnsfr9) is a member of the TNF-receptor (TNFR) superfamily without known intrinsic enzymatic activity in its cytoplasmic domain. Hence, akin to other members of the TNFR family, it relies on the TNFR-Associated-Factor (TRAF) family of adaptor proteins to build the CD137 signalosome for transducing signals into the cell. Thus, upon CD137 activation by binding of CD137L trimers or by crosslinking with agonist monoclonal antibodies, TRAF1, TRAF2, and TRAF3 are readily recruited to the cytoplasmic domain of CD137, likely as homo- and/or heterotrimers with different configurations, initiating the construction of the CD137 signalosome. The formation of TRAF2-RING dimers between TRAF2 molecules from contiguous trimers would help to establish a multimeric structure of TRAF-trimers that is probably essential for CD137 signaling. In addition, available studies have identified a large number of proteins that are recruited to CD137:TRAF complexes including ubiquitin ligases and proteases, kinases, and modulatory proteins. Working in a coordinated fashion, these CD137-signalosomes will ultimately promote CD137-mediated T cell proliferation and survival and will endow T cells with stronger effector functions. Current evidence allows to envision the molecular events that might take place in the early stages of CD137-signalosome formation, underscoring the key roles of TRAFs and of K63 and K48-ubiquitination of target proteins in the signaling process. Understanding the composition and fine regulation of CD137-signalosomes assembly and disassembly will be key to improve the therapeutic activities of chimeric antigen receptors (CARs) encompassing the CD137 cytoplasmic domain and a new generation of CD137 agonists for the treatment of cancer.

A kinetic investigation of interacting, stimulated T cells identifies conditions for rapid functional enhancement, minimal phenotype differentiation, and improved adoptive cell transfer tumor eradication.

For adoptive cell transfer (ACT) immunotherapy of tumor-reactive T cells, an effective therapeutic outcome depends upon cell dose, cell expansion in vivo through a minimally differentiated phenotype, long term persistence, and strong cytolytic effector function. An incomplete understanding of the biological coupling between T cell expansion, differentiation, and response to stimulation hinders the co-optimization of these factors. We report on a biophysical investigation of how the short-term kinetics of T cell functional activation, through molecular stimulation and cell-cell interactions, competes with phenotype differentiation. T cells receive molecular stimulation for a few minutes to a few hours in bulk culture. Following this priming period, the cells are then analyzed at the transcriptional level, or isolated as single cells, with continuing molecular stimulation, within microchambers for analysis via 11-plex secreted protein assays. We resolve a rapid feedback mechanism, promoted by T cell—T cell contact interactions, which strongly amplifies T cell functional performance while yielding only minimal phenotype differentiation. When tested in mouse models of ACT, optimally primed T cells lead to complete tumor eradication. A similar kinetic process is identified in CD8+ and CD4+ T cells collected from a patient with metastatic melanoma.

CD56 in the Immune System: More Than a Marker for Cytotoxicity?

Over the past years, the phenotypic and functional boundaries distinguishing the main cell subsets of the immune system have become increasingly blurred. In this respect, CD56 (also known as neural cell adhesion molecule) is a very good example. CD56 is the archetypal phenotypic marker of natural killer cells but can actually be expressed by many more immune cells, including alpha beta T cells, gamma delta T cells, dendritic cells, and monocytes. Common to all these CD56-expressing cell types are strong immunostimulatory effector functions, including T helper 1 cytokine production and an efficient cytotoxic capacity. Interestingly, both numerical and functional deficiencies and phenotypic alterations of the CD56+ immune cell fraction have been reported in patients with various infectious, autoimmune, or malignant diseases. In this review, we will discuss our current knowledge on the expression and function of CD56 in the hematopoietic system, both in health and disease.

Resident memory T cells in mouse and human.

Recent researches are revealing the importance of a new subset of memory T cells called resident memory T cells (TRMs). Once they enter the tissues according to their tissue-homing receptors, TRMs do not go back to circulation and stay in the same tissues for a long time. These T cells are defined as expressing CD69 and/or CD103, and are known to show strong effector functions. It is considered that TRMs have an important role against infection in barrier tissues such as GI tract, skin, respiratory system and reproductive tract. Furthermore, recent reports indicate their roles in organ-specific chronic inflammatory disorders, autoimmune disorders and tumor immunology even in non-barrier tissues such as central nerve system, lymphatic tissue, liver, kidney, pancreas and joint. Here in this session, the author organized what have been known about TRM both in mouse and human, including the development, functional activities and relation of TRM to disease manifestation, for the detailed understanding of this fraction.

Despite KLRG1 expression, highly cytotoxic ThEO/TcEO polarized T cells efficiently establish immunologic memory (LYM5P.662)

Abstract Targeting immune inhibitory (CTLA-4, PD-1) or stimulatory (OX-40, 4-1BB) co-receptors has proven a potent anti-cancer therapeutic strategy, eliciting robust anti-tumor T cell responses and compelling pre-clinical and clinical data in a variety of cancers. We have previously shown in pre-clinical models that 4-1BB agonist antibodies engender strong anti-tumor T cell responses, imparting enhanced survival benefit in addition to evoking durable tumor regression. This impressive anti-tumor effect is due, in part, to the unique ability of 4-1BB co-stimulation to differentiate a novel subset of T cells that acquires a highly cytotoxic effector profile typically only seen in NK cells. This subset-dubbed ThEO (CD4+) or TcEO (CD8+)-is driven by the T-box transcription factor Eomesodermin, and is further characterized by expression of the inhibitory receptor KLRG1. We show here that, despite their strong effector function and expression of several co-inhibitory receptors (PD-1, TIM-3, KLRG1), the ThEO/TcEO phenotype establishes immunological memory and recalls with high frequency upon secondary tumor challenge with potent anti-tumor effector function. Further, these cells manifest characteristics of central memory (Tcm), effector memory (Tem), as well as stem cell memory (Tscm), and reside in multiple memory niches. These findings provide mechanistic insight into the process of memory formation and give evidence for the capacity of ThEO/TcEO cells to prevent tumor recurrence.