PD-1 blockade does not improve efficacy of EpCAM-directed CAR T-cell in lung cancer brain metastasis.

BACKGROUND: Lung cancer brain metastasis has a devastating prognosis, necessitating innovative treatment strategies. While chimeric antigen receptor (CAR) T-cell show promise in hematologic malignancies, their efficacy in solid tumors, including brain metastasis, is limited by the immunosuppressive tumor environment. The PD-L1/PD-1 pathway inhibits CAR T-cell activity in the tumor microenvironment, presenting a potential target to enhance therapeutic efficacy. This study aims to evaluate the impact of anti-PD-1 antibodies on CAR T-cell in treating lung cancer brain metastasis. METHODS: We utilized a murine immunocompetent, syngeneic orthotopic cerebral metastasis model for repetitive intracerebral two-photon laser scanning microscopy, enabling in vivo characterization of red fluorescent tumor cells and CAR T-cell at a single-cell level over time. Red fluorescent EpCAM-transduced Lewis lung carcinoma cells (EpCAM/tdtLL/2 cells) were implanted intracranially. Following the formation of brain metastasis, EpCAM-directed CAR T-cell were injected into adjacent brain tissue, and animals received either anti-PD-1 or an isotype control. RESULTS: Compared to controls receiving T-cell lacking a CAR, mice receiving EpCAM-directed CAR T-cell showed higher intratumoral CAR T-cell densities in the beginning after intraparenchymal injection. This finding was accompanied with reduced tumor growth and translated into a survival benefit. Additional anti-PD-1 treatment, however, did not affect intratumoral CAR T-cell persistence nor tumor growth and thereby did not provide an additional therapeutic effect. CONCLUSION: CAR T-cell therapy for brain malignancies appears promising. However, additional anti-PD-1 treatment did not enhance intratumoral CAR T-cell persistence or effector function, highlighting the need for novel strategies to improve CAR T-cell therapy in solid tumors.

PGE2 limits effector expansion of tumour-infiltrating stem-like CD8+ T cells.

Cancer-specific TCF1+ stem-like CD8+ T cells can drive protective anticancer immunity through expansion and effector cell differentiation1-4; however, this response is dysfunctional in tumours. Current cancer immunotherapies2,5-9 can promote anticancer responses through TCF1+ stem-like CD8+ T cells in some but not all patients. This variation points towards currently ill-defined mechanisms that limit TCF1+CD8+ T cell-mediated anticancer immunity. Here we demonstrate that tumour-derived prostaglandin E2 (PGE2) restricts the proliferative expansion and effector differentiation of TCF1+CD8+ T cells within tumours, which promotes cancer immune escape. PGE2 does not affect the priming of TCF1+CD8+ T cells in draining lymph nodes. PGE2 acts through EP2 and EP4 (EP2/EP4) receptor signalling in CD8+ T cells to limit the intratumoural generation of early and late effector T cell populations that originate from TCF1+ tumour-infiltrating CD8+ T lymphocytes (TILs). Ablation of EP2/EP4 signalling in cancer-specific CD8+ T cells rescues their expansion and effector differentiation within tumours and leads to tumour elimination in multiple mouse cancer models. Mechanistically, suppression of the interleukin-2 (IL-2) signalling pathway underlies the PGE2-mediated inhibition of TCF1+ TIL responses. Altogether, we uncover a key mechanism that restricts the IL-2 responsiveness of TCF1+ TILs and prevents anticancer T cell responses that originate from these cells. This study identifies the PGE2-EP2/EP4 axis as a molecular target to restore IL-2 responsiveness in anticancer TILs to achieve cancer immune control.

B cells orchestrate tolerance to the neuromyelitis optica autoantigen AQP4.

Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen1. The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP42. However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.

A distinct stimulatory cDC1 subpopulation amplifies CD8+ T cell responses in tumors for protective anti-cancer immunity.

Type 1 conventional dendritic cells (cDC1) can support T cell responses within tumors but whether this determines protective versus ineffective anti-cancer immunity is poorly understood. Here, we use imaging-based deep learning to identify intratumoral cDC1-CD8+ T cell clustering as a unique feature of protective anti-cancer immunity. These clusters form selectively in stromal tumor regions and constitute niches in which cDC1 activate TCF1+ stem-like CD8+ T cells. We identify a distinct population of immunostimulatory CCR7neg cDC1 that produce CXCL9 to promote cluster formation and cross-present tumor antigens within these niches, which is required for intratumoral CD8+ T cell differentiation and expansion and promotes cancer immune control. Similarly, in human cancers, CCR7neg cDC1 interact with CD8+ T cells in clusters and are associated with patient survival. Our findings reveal an intratumoral phase of the anti-cancer T cell response orchestrated by tumor-residing cDC1 that determines protective versus ineffective immunity and could be exploited for cancer therapy.

Tumor-derived prostaglandin E2 programs cDC1 dysfunction to impair intratumoral orchestration of anti-cancer T cell responses.

Type 1 conventional dendritic cells (cDC1s) are critical for anti-cancer immunity. Protective anti-cancer immunity is thought to require cDC1s to sustain T cell responses within tumors, but it is poorly understood how this function is regulated and whether its subversion contributes to immune evasion. Here, we show that tumor-derived prostaglandin E2 (PGE2) programmed a dysfunctional state in intratumoral cDC1s, disabling their ability to locally orchestrate anti-cancer CD8+ T cell responses. Mechanistically, cAMP signaling downstream of the PGE2-receptors EP2 and EP4 was responsible for the programming of cDC1 dysfunction, which depended on the loss of the transcription factor IRF8. Blockade of the PGE2-EP2/EP4-cDC1 axis prevented cDC1 dysfunction in tumors, locally reinvigorated anti-cancer CD8+ T cell responses, and achieved cancer immune control. In human cDC1s, PGE2-induced dysfunction is conserved and associated with poor cancer patient prognosis. Our findings reveal a cDC1-dependent intratumoral checkpoint for anti-cancer immunity that is targeted by PGE2 for immune evasion.

Unbiased chemokine receptor screening reveals similar efficacy of lymph node- and tumor-targeted T cell immunotherapy.

Localization is a crucial prerequisite for immune cell function and solid tumors evade immune control by modulating immune cell infiltration into the tumor stroma. Immunosuppressive cells like regulatory T cells are attracted, while cytotoxic CD8+ T cells are excluded. Engineering CD8+ T cells with chemokine receptors is a potent strategy to turn this mechanism of directed immune cell recruitment against the tumor. Here, we utilized fluorescent tagging to track the migratory behavior of tumor-specific T cells engineered with a library of all murine chemokine receptors in vivo. We then asked whether chemokine receptor-mediated redirection of antigen-specific T cells into tumors or tumor-draining lymph nodes showed superior anti-tumoral activity. We found that both targeting approaches showed higher therapeutic efficacy than control T cells. However, multiple receptors conveying the same homing pattern did not augment infiltration. Instead, in the MC38 colon carcinoma model, anti-tumoral efficacy as well as lymph node vs. tumor-homing patterns were mostly driven by CCR4 and CCR6, respectively. Overall, our data, based on fluorescent receptor tagging, identify the tumor-draining lymph node and the tumor itself as viable targets for chemokine receptor-mediated enhancement of adoptive T cell therapy.

In vivo dynamics and anti-tumor effects of EpCAM-directed CAR T-cells against brain metastases from lung cancer.

Lung cancer patients are at risk for brain metastases and often succumb to their intracranial disease. Chimeric Antigen Receptor (CAR) T-cells emerged as a powerful cell-based immunotherapy for hematological malignancies; however, it remains unclear whether CAR T-cells represent a viable therapy for brain metastases. Here, we established a syngeneic orthotopic cerebral metastasis model in mice by combining a chronic cranial window with repetitive intracerebral two-photon laser scanning-microscopy. This approach enabled in vivo-characterization of fluorescent CAR T-cells and tumor cells on a single-cell level over weeks. Intraparenchymal injection of Lewis lung carcinoma cells (expressing the tumor cell-antigen EpCAM) was performed, and EpCAM-directed CAR T-cells were injected either intravenously or into the adjacent brain parenchyma. In mice receiving EpCAM-directed CAR T-cells intravenously, we neither observed substantial CAR T-cell accumulation within the tumor nor relevant anti-tumor effects. Local CAR T-cell injection, however, resulted in intratumoral CAR T-cell accumulation compared to controls treated with T-cells lacking a CAR. This finding was accompanied by reduced tumorous growth as determined per in vivo-microscopy and immunofluorescence of excised brains and also translated into prolonged survival. However, the intratumoral number of EpCAM-directed CAR T-cells decreased during the observation period, pointing toward insufficient persistence. No CNS-specific or systemic toxicities of EpCAM-directed CAR T-cells were observed in our fully immunocompetent model. Collectively, our findings indicate that locally (but not intravenously) injected CAR T-cells may safely induce relevant anti-tumor effects in brain metastases from lung cancer. Strategies improving the intratumoral CAR T-cell persistence may further boost the therapeutic success.

CagA-specific Gastric CD8+ Tissue-Resident T Cells Control Helicobacter pylori During the Early Infection Phase.

BACKGROUND & AIMS: Infection with Helicobacter pylori strongly affects global health by causing chronic gastritis, ulcer disease, and gastric cancer. Although extensive research into the strong immune response against this persistently colonizing bacterium exists, the specific role of CD8+ T cells remains elusive. METHODS: We comprehensively characterize gastric H pylori-specific CD8+ T-cell responses in mice and humans by flow cytometry, RNA-sequencing, immunohistochemistry, and ChipCytometry, applying functional analyses including T-cell depletion, H pylori eradication, and ex vivo restimulation. RESULTS: We define CD8+ T-cell populations bearing a tissue-resident memory (TRM) phenotype, which infiltrate the gastric mucosa shortly after infection and mediate pathogen control by executing antigen-specific effector properties. These induced CD8+ tissue-resident memory T cells (TRM cells) show a skewed T-cell receptor beta chain usage and are mostly specific for cytotoxin-associated gene A, the distinctive oncoprotein injected by H pylori into host cells. As the infection progresses, we observe a loss of the TRM phenotype and replacement of CD8+ by CD4+ T cells, indicating a shift in the immune response during the chronic infection phase. CONCLUSIONS: Our results point toward a hitherto unknown role of CD8+ T-cell response in this bacterial infection, which may have important clinical implications for treatment and vaccination strategies against H pylori.

MYB orchestrates T cell exhaustion and response to checkpoint inhibition.

CD8+ T cells that respond to chronic viral infections or cancer are characterized by the expression of inhibitory receptors such as programmed cell death protein 1 (PD-1) and by the impaired production of cytokines. This state of restrained functionality-which is referred to as T cell exhaustion1,2-is maintained by precursors of exhausted T (TPEX) cells that express the transcription factor T cell factor 1 (TCF1), self-renew and give rise to TCF1- exhausted effector T cells3-6. Here we show that the long-term proliferative potential, multipotency and repopulation capacity of exhausted T cells during chronic infection are selectively preserved in a small population of transcriptionally distinct CD62L+ TPEX cells. The transcription factor MYB is not only essential for the development of CD62L+ TPEX cells and maintenance of the antiviral CD8+ T cell response, but also induces functional exhaustion and thereby prevents lethal immunopathology. Furthermore, the proliferative burst in response to PD-1 checkpoint inhibition originates exclusively from CD62L+ TPEX cells and depends on MYB. Our findings identify CD62L+ TPEX cells as a stem-like population that is central to the maintenance of long-term antiviral immunity and responsiveness to immunotherapy. Moreover, they show that MYB is a transcriptional orchestrator of two fundamental aspects of exhausted T cell responses: the downregulation of effector function and the long-term preservation of self-renewal capacity.

Signatures of recent activation identify a circulating T cell compartment containing tumor-specific antigen receptors with high avidity.

T cell receptor (TCR) avidity is assumed to be a major determinant of the spatiotemporal fate and protective capacity of tumor-specific T cells. However, monitoring polyclonal T cell responses with known TCR avidities in vivo over space and time remains challenging. Here, we investigated the fate and functionality of tumor neoantigen-specific T cells with TCRs of distinct avidities in a well-established, reductionist preclinical tumor model and human patients with melanoma. To this end, we used polyclonal T cell transfers with in-depth characterized TCRs together with flow cytometric phenotyping in mice inoculated with MC38 OVA tumors. Transfer of T cells from retrogenic mice harboring TCRs with high avidity resulted in best tumor protection. Unexpectedly, we found that both high- and low-avidity T cells are similarly abundant within the tumor and adopt concordant phenotypic signs of exhaustion. Outside the tumor, high-avidity TCR T cells were not generally overrepresented but, instead, selectively enriched in T cell populations with intermediate PD-1 protein expression. Single-cell sequencing of neoantigen-specific T cells from two patients with melanoma-combined with transgenic reexpression of identified TCRs by CRISPR-Cas9-mediated orthotopic TCR replacement-revealed high-functionality TCRs to be enriched in T cells with RNA signatures of recent activation. Furthermore, of 130 surface protein candidates, PD-1 surface expression was most consistently enriched in functional TCRs. Together, our findings show that tumor-reactive TCRs with high protective capacity circulating in peripheral blood are characterized by a signature of recent activation.

Retrogenic Color-Barcoding for Fate Mapping of Single Innate Lymphocytes.

Lymphocyte fate mapping using single-cell transfers has been used to study T and B cell differentiation. Recently, retrogenic color-barcoding has allowed the extension of this approach to single innate lymphocytes such as NK cells. This new and versatile technology is based on the transduction of hematopoietic stem cells (HSCs) with a collection of retroviruses encoding distinct fluorescent proteins. Through combinatorial transduction, fluorescent protein barcodes are generated, which are inherited by the progeny of HSCs after transfer. By sorting individual cells expressing unique color-barcodes from the mature lymphocyte populations derived from these HSCs, it is now possible to track the fate of innate lymphocytes in vivo.

Early emergence of T central memory precursors programs clonal dominance during chronic viral infection.

Chronic cytomegalovirus (CMV) infection leads to long-term maintenance of extraordinarily large CMV-specific T cell populations. The magnitude of this so-called 'memory inflation' is thought to mainly depend on antigenic stimulation during the chronic phase of infection. However, by mapping the long-term development of CD8+ T cell families derived from single naive precursors, we find that fate decisions made during the acute phase of murine CMV infection can alter the level of memory inflation by more than 1,000-fold. Counterintuitively, a T cell family's capacity for memory inflation is not determined by its initial expansion. Instead, those rare T cell families that dominate the chronic phase of infection show an early transcriptomic signature akin to that of established T central memory cells. Accordingly, a T cell family's long-term dominance is best predicted by its early content of T central memory precursors, which later serve as a stem-cell-like source for memory inflation.

Multiplexed whole-animal imaging with reversibly switchable optoacoustic proteins.

We introduce two photochromic proteins for cell-specific in vivo optoacoustic (OA) imaging with signal unmixing in the temporal domain. We show highly sensitive, multiplexed visualization of T lymphocytes, bacteria, and tumors in the mouse body and brain. We developed machine learning-based software for commercial imaging systems for temporal unmixed OA imaging, enabling its routine use in life sciences.

Long-term in vivo microscopy of CAR T cell dynamics during eradication of CNS lymphoma in mice.

T cells expressing anti-CD19 chimeric antigen receptors (CARs) demonstrate impressive efficacy in the treatment of systemic B cell malignancies, including B cell lymphoma. However, their effect on primary central nervous system lymphoma (PCNSL) is unknown. Additionally, the detailed cellular dynamics of CAR T cells during their antitumor reaction remain unclear, including their intratumoral infiltration depth, mobility, and persistence. Studying these processes in detail requires repeated intravital imaging of precisely defined tumor regions during weeks of tumor growth and regression. Here, we have combined a model of PCNSL with in vivo intracerebral 2-photon microscopy. Thereby, we were able to visualize intracranial PCNSL growth and therapeutic effects of CAR T cells longitudinally in the same animal over several weeks. Intravenous (i.v.) injection resulted in poor tumor infiltration of anti-CD19 CAR T cells and could not sufficiently control tumor growth. After intracerebral injection, however, anti-CD19 CAR T cells invaded deeply into the solid tumor, reduced tumor growth, and induced regression of PCNSL, which was associated with long-term survival. Intracerebral anti-CD19 CAR T cells entered the circulation and infiltrated distant, nondraining lymph nodes more efficiently than mock CAR T cells. After complete regression of tumors, anti-CD19 CAR T cells remained detectable intracranially and intravascularly for up to 159 d. Collectively, these results demonstrate the great potential of anti-CD19 CAR T cells for the treatment of PCNSL.

TCR repertoire evolution during maintenance of CMV-specific T-cell populations.

During infections and cancer, the composition of the T-cell receptor (TCR) repertoire of antigen-specific CD8+ T cells changes over time. TCR avidity is thought to be a major driver of this process, thereby interacting with several additional regulators of T-cell responses to form a composite immune response architecture. Infections with latent viruses, such as cytomegalovirus (CMV), can lead to large T-cell responses characterized by an oligoclonal TCR repertoire. Here, we review the current status of experimental studies and theoretical models of TCR repertoire evolution during CMV infection. We will particularly discuss the degree to which this process may be determined through structural TCR avidity. As engineered TCR-redirected T cells have moved into the spotlight for providing more effective immunotherapies, it is essential to understand how the key features of a given TCR influence T-cell expansion and maintenance in settings of infection or malignancy. Deeper insights into these mechanisms will improve our basic understanding of T-cell immunology and help to identify optimal TCRs for immunotherapy.

Role of memory T cell subsets for adoptive immunotherapy.

Adoptive transfer of primary (unmodified) or genetically engineered antigen-specific T cells has demonstrated astonishing clinical results in the treatment of infections and some malignancies. Besides the definition of optimal targets and antigen receptors, the differentiation status of transferred T cells is emerging as a crucial parameter for generating cell products with optimal efficacy and safety profiles. Long-living memory T cells subdivide into phenotypically as well as functionally different subsets (e.g. central memory, effector memory, tissue-resident memory T cells). This diversification process is crucial for effective immune protection, with probably distinct dependencies on the presence of individual subsets dependent on the disease to which the immune response is directed as well as its organ location. Adoptive T cell therapy intends to therapeutically transfer defined T cell immunity into patients. Efficacy of this approach often requires long-term maintenance of transferred cells, which depends on the presence and persistence of memory T cells. However, engraftment and survival of highly differentiated memory T cell subsets upon adoptive transfer is still difficult to achieve. Therefore, the recent observation that a distinct subset of weakly differentiated memory T cells shows all characteristics of adult tissue stem cells and can reconstitute all types of effector and memory T cell subsets, became highly relevant. We here review our current understanding of memory subset formation and T cell subset purification, and its implications for adoptive immunotherapy.

Serial transfer of single-cell-derived immunocompetence reveals stemness of CD8(+) central memory T cells.

Maintenance of immunological memory has been proposed to rely on stem-cell-like lymphocytes. However, data supporting this hypothesis are focused on the developmental potential of lymphocyte populations and are thus insufficient to establish the functional hallmarks of stemness. Here, we investigated self-renewal capacity and multipotency of individual memory lymphocytes by in vivo fate mapping of CD8(+) T cells and their descendants across three generations of serial single-cell adoptive transfer and infection-driven re-expansion. We found that immune responses derived from single naive T (Tn) cells, single primary, and single secondary central memory T (Tcm) cells reached similar size and phenotypic diversity, were subjected to comparable stochastic variation, and could ultimately reconstitute immunocompetence against an otherwise lethal infection with the bacterial pathogen Listeria monocytogenes. These observations establish that adult tissue stem cells reside within the CD62L(+) Tcm cell compartment and highlight the promising therapeutic potential of this immune cell subset.

Lowest numbers of primary CD8(+) T cells can reconstitute protective immunity upon adoptive immunotherapy.

Patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are threatened by potentially lethal viral manifestations like cytomegalovirus (CMV) reactivation. Because the success of today's virostatic treatment is limited by side effects and resistance development, adoptive transfer of virus-specific memory T cells derived from the stem cell donor has been proposed as an alternative therapeutic strategy. In this context, dose minimization of adoptively transferred T cells might be warranted for the avoidance of graft-versus-host disease (GVHD), in particular in prophylactic settings after T-cell-depleting allo-HSCT protocols. To establish a lower limit for successful adoptive T-cell therapy, we conducted low-dose CD8(+) T-cell transfers in the well-established murine Listeria monocytogenes (L.m.) infection model. Major histocompatibility complex-Streptamer-enriched antigen-specific CD62L(hi) but not CD62L(lo) CD8(+) memory T cells proliferated, differentiated, and protected against L.m. infections after prophylactic application. Even progenies derived from a single CD62L(hi) L.m.-specific CD8(+) T cell could be protective against bacterial challenge. In analogy, low-dose transfers of Streptamer-enriched human CMV-specific CD8(+) T cells into allo-HSCT recipients led to strong pathogen-specific T-cell expansion in a compassionate-use setting. In summary, low-dose adoptive T-cell transfer (ACT) could be a promising strategy, particularly for prophylactic treatment of infectious complications after allo-HSCT.

Antigen delivery to CD11c+CD8- dendritic cells induces protective immune responses against experimental melanoma in mice in vivo.

Dendritic cells (DCs) are central modulators of immune responses and, therefore, interesting target cells for the induction of antitumor immune responses. Ag delivery to select DC subpopulations via targeting Abs to DC inhibitory receptor 2 (DCIR2, clone 33D1) or to DEC205 was shown to direct Ags specifically to CD11c(+)CD8(-) or CD11c(+)CD8(+) DCs, respectively, in vivo. In contrast to the increasing knowledge about the induction of immune responses by efficiently cross-presenting CD11c(+)CD8(+) DCs, little is known about the functional role of Ag-presenting CD11c(+)CD8(-) DCs with regard to the initiation of protective immune responses. In this study, we demonstrate that Ag targeting to the CD11c(+)CD8(-) DC subpopulation in the presence of stimulating anti-CD40 Ab and TLR3 ligand polyinosinic-polycytidylic acid induces protective responses against rapidly growing tumor cells in naive animals under preventive and therapeutic treatment regimens in vivo. Of note, this immunization protocol induced a mixed Th1/Th2-driven immune response, irrespective of which DC subpopulation initially presented the Ag. Our results provide important information about the role of CD11c(+)CD8(-) DCs, which have been considered to be less efficient at cross-presenting Ags, in the induction of protective antitumor immune responses.

The smallest unit: effector and memory CD8(+) T cell differentiation on the single cell level.

CD8(+) T cell immune responses provide immediate protection against primary infection and durable memory capable of rapidly fighting off re-infection. Immediate protection and lasting memory are implemented by phenotypically and functionally distinct T cell subsets. While it is now widely accepted that these diverge from a common source of naïve T cells (T(n)), the developmental relation and succession of effector and memory T cell subsets is still under intense debate. Recently, a distinct memory T cell subset has been suggested to possess stem cell-like features, sparking the hope to harness its capacity for self-renewal and diversification for successful therapy of chronic infections or malignant diseases. In this review we highlight current developmental models of memory generation, T cell subset diversification and T cell stemness. We discuss the importance of single cell monitoring techniques for adequately mapping these developmental processes and take a brief look at signaling components active in the putative stem cell-like memory T cell compartment.