Synthetic soldiers: Turning T cells into immortal warriors.

The creation of synthetic T cell states has captivated the field of cell-based therapies. Wang et al. (https://doi.org/10.1084/jem.20232368) describe how disruption of BCOR and ZC3H12A unleashes anti-tumor T cells with unprecedented lifespan and killer instinct. Are we witnessing the birth of immortal super-soldiers in medicine?

Rapid Screening of CAR T Cell Functional Improvement Strategies by Highly Multiplexed Single-Cell Secretomics.

The functional fitness of CAR T cells plays a crucial role in determining their clinical efficacy. Several strategies are being explored to increase cellular fitness, but screening these approaches in vivo is expensive and time-consuming, limiting the number of strategies that can be tested at one time. The presence of polyfunctional CAR T cells has emerged as a critical parameter correlating with clinical responses. However, even sophisticated multiplexed secretomic assays often fail to detect differences in cytokine release due to the functional heterogeneity of CAR T cell products. Here, we describe a highly multiplexed single-cell secretomic assay based on the IsoLight platform to rapidly evaluate the impact of new pharmacologic or gene-engineering approaches aiming at improving CAR T cell function. As a key study, we focus on CD19-specific CAR CD8+ T cells modulated by miR-155 overexpression, but the protocol can be applied to characterize other functional immune cell modulation strategies.

Releasing the brake: CTLA-4 loss turbocharges CAR T cells.

Immune checkpoint receptor-induced T cell dysfunction is a major cause of CAR T cell treatment failure. In this issue, Agarwal et al. report that CRISPR/Cas9 deletion of CTLA4, but not PDCD1 or CTLA4 and PDCD1, enhances CD28 signaling, restoring fitness and antitumor function of CAR T cells, including those derived from patients who failed CAR T cell therapy.

Targeting lactate metabolism for cancer immunotherapy - a matter of precision.

Immune checkpoint inhibitors and adoptive T cell therapies have been valuable additions to the toolbox in the fight against cancer. These treatments have profoundly increased the number of patients with a realistic perspective toward a return to a cancer-free life. Yet, in a number of patients and tumor entities, cancer immunotherapies have been ineffective so far. In solid tumors, immune exclusion and the immunosuppressive tumor microenvironment represent substantial roadblocks to successful therapeutic outcomes. A major contributing factor to the depressed anti-tumor activity of immune cells in tumors is the harsh metabolic environment. Hypoxia, nutrient competition with tumor and stromal cells, and accumulating noxious waste products, including lactic acid, pose massive constraints to anti-tumor immune cells. Numerous strategies are being developed to exploit the metabolic vulnerabilities of tumor cells in the hope that these would also alleviate metabolism-inflicted immune suppression. While promising in principle, especially in combination with immunotherapies, these strategies need to be scrutinized for their effect on tumor-fighting immune cells, which share some of their key metabolic properties with tumor cells. Here, we provide an overview of strategies that seek to tackle lactate metabolism in tumor or immune cells to unleash anti-tumor immune responses, thereby opening therapeutic options for patients whose tumors are currently not treatable.

The New Old CD8+ T Cells in the Immune Paradox of Pregnancy.

CD8+ T cells are the most frequent T cell population in the immune cell compartment at the feto-maternal interface. Due to their cytotoxic potential, the presence of CD8+ T cells in the immune privileged pregnant uterus has raised considerable interest. Here, we review our current understanding of CD8+ T cell biology in the uterus of pregnant women and discuss this knowledge in relation to a recently published immune cell Atlas of human decidua. We describe how the expansion of CD8+ T cells with an effector memory phenotype often presenting markers of exhaustion is critical for a successful pregnancy, and host defense towards pathogens. Moreover, we review new evidence on the presence of long-lasting immunological memory to former pregnancies and discuss its impact on prospective pregnancy outcomes. The formation of fetal-specific memory CD8+ T cell subests in the uterus, in particular of tissue resident, and stem cell memory cells requires further investigation, but promises interesting results to come. Advancing the knowledge of CD8+ T cell biology in the pregnant uterus will be pivotal for understanding not only tissue-specific immune tolerance but also the etiology of complications during pregnancy, thus enabling preventive or therapeutic interventions in the future.

An engineered IL-2 partial agonist promotes CD8+ T cell stemness.

Adoptive transfer of antigen-specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in some patients1. Both the number of transferred T cells and their differentiation state are critical determinants of effective responses2,3. T cells can be expanded with T cell receptor (TCR)-mediated stimulation and interleukin-2, but this can lead to differentiation into effector T cells4,5 and lower therapeutic efficacy6, whereas maintenance of a more stem-cell-like state before adoptive transfer is beneficial7. Here we show that H9T, an engineered interleukin-2 partial agonist, promotes the expansion of CD8+ T cells without driving terminal differentiation. H9T led to altered STAT5 signalling and mediated distinctive downstream transcriptional, epigenetic and metabolic programs. In addition, H9T treatment sustained the expression of T cell transcription factor 1 (TCF-1) and promoted mitochondrial fitness, thereby facilitating the maintenance of a stem-cell-like state. Moreover, TCR-transgenic and chimeric antigen receptor-modified CD8+ T cells that were expanded with H9T showed robust anti-tumour activity in vivo in mouse models of melanoma and acute lymphoblastic leukaemia. Thus, engineering cytokine variants with distinctive properties is a promising strategy for creating new molecules with translational potential.

BACH2 enforces the transcriptional and epigenetic programs of stem-like CD8+ T cells.

During chronic infection and cancer, a self-renewing CD8+ T cell subset maintains long-term immunity and is critical to the effectiveness of immunotherapy. These stem-like CD8+ T cells diverge from other CD8+ subsets early after chronic viral infection. However, pathways guarding stem-like CD8+ T cells against terminal exhaustion remain unclear. Here, we show that the gene encoding transcriptional repressor BACH2 is transcriptionally and epigenetically active in stem-like CD8+ T cells but not terminally exhausted cells early after infection. BACH2 overexpression enforced stem-like cell fate, whereas BACH2 deficiency impaired stem-like CD8+ T cell differentiation. Single-cell transcriptomic and epigenomic approaches revealed that BACH2 established the transcriptional and epigenetic programs of stem-like CD8+ T cells. In addition, BACH2 suppressed the molecular program driving terminal exhaustion through transcriptional repression and epigenetic silencing. Thus, our study reveals a new pathway that enforces commitment to stem-like CD8+ lineage and prevents an alternative terminally exhausted cell fate.

Two subsets of stem-like CD8+ memory T cell progenitors with distinct fate commitments in humans.

T cell memory relies on the generation of antigen-specific progenitors with stem-like properties. However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells. We used a systematic approach guided by single-cell RNA-sequencing data to map the organizational structure of the human CD8+ memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically and transcriptionally distinct stem-like CD8+ memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage. Collectively, these data reveal the existence of parallel differentiation programs in the human CD8+ memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines.

Lactate dehydrogenase inhibition synergizes with IL-21 to promote CD8+ T cell stemness and antitumor immunity.

Interleukin (IL)-2 and IL-21 dichotomously shape CD8+ T cell differentiation. IL-2 drives terminal differentiation, generating cells that are poorly effective against tumors, whereas IL-21 promotes stem cell memory T cells (TSCM) and antitumor responses. Here we investigated the role of metabolic programming in the developmental differences induced by these cytokines. IL-2 promoted effector-like metabolism and aerobic glycolysis, robustly inducing lactate dehydrogenase (LDH) and lactate production, whereas IL-21 maintained a metabolically quiescent state dependent on oxidative phosphorylation. LDH inhibition rewired IL-2-induced effects, promoting pyruvate entry into the tricarboxylic acid cycle and inhibiting terminal effector and exhaustion programs, including mRNA expression of members of the NR4A family of nuclear receptors, as well as Prdm1 and Xbp1 While deletion of Ldha prevented development of cells with antitumor effector function, transient LDH inhibition enhanced the generation of memory cells capable of triggering robust antitumor responses after adoptive transfer. LDH inhibition did not significantly affect IL-21-induced metabolism but caused major transcriptomic changes, including the suppression of IL-21-induced exhaustion markers LAG3, PD1, 2B4, and TIM3. LDH inhibition combined with IL-21 increased the formation of TSCM cells, resulting in more profound antitumor responses and prolonged host survival. These findings indicate a pivotal role for LDH in modulating cytokine-mediated T cell differentiation and underscore the therapeutic potential of transiently inhibiting LDH during adoptive T cell-based immunotherapy, with an unanticipated cooperative antitumor effect of LDH inhibition and IL-21.

CXCR3 Identifies Human Naive CD8+ T Cells with Enhanced Effector Differentiation Potential.

In mice, the ability of naive T (TN) cells to mount an effector response correlates with TCR sensitivity for self-derived Ags, which can be quantified indirectly by measuring surface expression levels of CD5. Equivalent findings have not been reported previously in humans. We identified two discrete subsets of human CD8+ TN cells, defined by the absence or presence of the chemokine receptor CXCR3. The more abundant CXCR3+ TN cell subset displayed an effector-like transcriptional profile and expressed TCRs with physicochemical characteristics indicative of enhanced interactions with peptide-HLA class I Ags. Moreover, CXCR3+ TN cells frequently produced IL-2 and TNF in response to nonspecific activation directly ex vivo and differentiated readily into Ag-specific effector cells in vitro. Comparative analyses further revealed that human CXCR3+ TN cells were transcriptionally equivalent to murine CXCR3+ TN cells, which expressed high levels of CD5. These findings provide support for the notion that effector differentiation is shaped by heterogeneity in the preimmune repertoire of human CD8+ T cells.

Single-cell RNA-seq reveals TOX as a key regulator of CD8+ T cell persistence in chronic infection.

Progenitor-like CD8+ T cells mediate long-term immunity to chronic infection and cancer and respond potently to immune checkpoint blockade. These cells share transcriptional regulators with memory precursor cells, including T cell-specific transcription factor 1 (TCF1), but it is unclear whether they adopt distinct programs to adapt to the immunosuppressive environment. By comparing the single-cell transcriptomes and epigenetic profiles of CD8+ T cells responding to acute and chronic viral infections, we found that progenitor-like CD8+ T cells became distinct from memory precursor cells before the peak of the T cell response. We discovered a coexpression gene module containing Tox that exhibited higher transcriptional activity associated with more abundant active histone marks in progenitor-like cells than memory precursor cells. Moreover, thymocyte selection-associated high mobility group box protein TOX (TOX) promoted the persistence of antiviral CD8+ T cells and was required for the programming of progenitor-like CD8+ T cells. Thus, long-term CD8+ T cell immunity to chronic viral infection requires unique transcriptional and epigenetic programs associated with the transcription factor TOX.

miR-155 harnesses Phf19 to potentiate cancer immunotherapy through epigenetic reprogramming of CD8+ T cell fate.

T cell senescence and exhaustion are major barriers to successful cancer immunotherapy. Here we show that miR-155 increases CD8+ T cell antitumor function by restraining T cell senescence and functional exhaustion through epigenetic silencing of drivers of terminal differentiation. miR-155 enhances Polycomb repressor complex 2 (PRC2) activity indirectly by promoting the expression of the PRC2-associated factor Phf19 through downregulation of the Akt inhibitor, Ship1. Phf19 orchestrates a transcriptional program extensively shared with miR-155 to restrain T cell senescence and sustain CD8+ T cell antitumor responses. These effects rely on Phf19 histone-binding capacity, which is critical for the recruitment of PRC2 to the target chromatin. These findings establish the miR-155-Phf19-PRC2 as a pivotal axis regulating CD8+ T cell differentiation, thereby paving new ways for potentiating cancer immunotherapy through epigenetic reprogramming of CD8+ T cell fate.

The transcription factor c-Myb regulates CD8+ T cell stemness and antitumor immunity.

Stem cells are maintained by transcriptional programs that promote self-renewal and repress differentiation. Here, we found that the transcription factor c-Myb was essential for generating and maintaining stem cells in the CD8+ T cell memory compartment. Following viral infection, CD8+ T cells lacking Myb underwent terminal differentiation and generated fewer stem cell-like central memory cells than did Myb-sufficient T cells. c-Myb acted both as a transcriptional activator of Tcf7 (which encodes the transcription factor Tcf1) to enhance memory development and as a repressor of Zeb2 (which encodes the transcription factor Zeb2) to hinder effector differentiation. Domain-mutagenesis experiments revealed that the transactivation domain of c-Myb was necessary for restraining differentiation, whereas its negative regulatory domain was critical for cell survival. Myb overexpression enhanced CD8+ T cell memory formation, polyfunctionality and recall responses that promoted curative antitumor immunity after adoptive transfer. These findings identify c-Myb as a pivotal regulator of CD8+ T cell stemness and highlight its therapeutic potential.

T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy.

Across clinical trials, T cell expansion and persistence following adoptive cell transfer (ACT) have correlated with superior patient outcomes. Herein, we undertook a pan-cancer analysis to identify actionable ligand-receptor pairs capable of compromising T cell durability following ACT. We discovered that FASLG, the gene encoding the apoptosis-inducing ligand FasL, is overexpressed within the majority of human tumor microenvironments (TMEs). Further, we uncovered that Fas, the receptor for FasL, is highly expressed on patient-derived T cells used for clinical ACT. We hypothesized that a cognate Fas-FasL interaction within the TME might limit both T cell persistence and antitumor efficacy. We discovered that genetic engineering of Fas variants impaired in the ability to bind FADD functioned as dominant negative receptors (DNRs), preventing FasL-induced apoptosis in Fas-competent T cells. T cells coengineered with a Fas DNR and either a T cell receptor or chimeric antigen receptor exhibited enhanced persistence following ACT, resulting in superior antitumor efficacy against established solid and hematologic cancers. Despite increased longevity, Fas DNR-engineered T cells did not undergo aberrant expansion or mediate autoimmunity. Thus, T cell-intrinsic disruption of Fas signaling through genetic engineering represents a potentially universal strategy to enhance ACT efficacy across a broad range of human malignancies.

Ex vivo AKT-inhibition facilitates generation of polyfunctional stem cell memory-like CD8+ T cells for adoptive immunotherapy.

Adoptive T cell therapy has shown clinical potential for patients with cancer, though effective treatment is dependent on longevity and potency of the exploited tumor-reactive T cells. Previously, we showed that ex vivo inhibition of AKT using the research compound Akt-inhibitor VIII retained differentiation and improved functionality of minor histocompatibility antigen (MiHA)-specific CD8+ T cells. Here, we compared a panel of clinically applicable AKT-inhibitors with an allosteric or adenosine triphosphate-competitive mode of action. We analyzed phenotype, functionality, metabolism and transcriptome of AKT-inhibited CD8+ T cells using different T cell activation models. Most inhibitors facilitated T cell expansion while preserving an early memory phenotype, reflected by maintenance of CD62L, CCR7 and CXCR4 expression. Moreover, transcriptome profiling revealed that AKT-inhibited CD8+ T cells clustered closely to naturally occurring stem cell-memory CD8+ T cells, while control T cells resembled effector-memory T cells. Interestingly, AKT-inhibited CD8+ T cells showed enrichment of hypoxia-associated genes, which was consistent with enhanced glycolytic function. Notably, AKT-inhibition during MiHA-specific CD8+ T cell priming uncoupled preservation of early memory differentiation from ex vivo expansion. Furthermore, AKT-inhibited MiHA-specific CD8+ T cells showed increased polyfunctionality with co-secretion of IFN-γ and IL-2 upon antigen recall. Together, these data demonstrate that AKT-inhibitors with different modality of action promote the ex vivo generation of stem cell memory-like CD8+ T cells with a unique metabolic profile and retained polyfunctionality. Akt-inhibitor VIII and GDC-0068 outperformed other inhibitors, and are therefore promising candidates for ex vivo generation of superior tumor-reactive T cells for adoptive immunotherapy in cancer patients.