Multimodal single-cell profiling of intrahepatic cholangiocarcinoma defines hyperactivated Tregs as a potential therapeutic target.

BACKGROUND & AIMS: The landscape and function of the immune infiltrate of intrahepatic cholangiocarcinoma (iCCA), a rare, yet aggressive tumor of the biliary tract, remains poorly characterized, limiting development of successful immunotherapies. Herein, we aimed to define the molecular characteristics of tumor-infiltrating leukocytes with a special focus on CD4+ regulatory T cells (Tregs). METHODS: We used high-dimensional single-cell technologies to characterize the T-cell and myeloid compartments of iCCA tissues, comparing these with their tumor-free peritumoral and circulating counterparts. We further used genomics and cellular assays to define the iCCA-specific role of a novel transcription factor, mesenchyme homeobox 1 (MEOX1), in Treg biology. RESULTS: We found poor infiltration of putative tumor-specific CD39+ CD8+ T cells accompanied by abundant infiltration of hyperactivated CD4+ Tregs. Single-cell RNA-sequencing identified an altered network of transcription factors in iCCA-infiltrating compared to peritumoral T cells, suggesting reduced effector functions by tumor-infiltrating CD8+ T cells and enhanced immunosuppression by CD4+ Tregs. Specifically, we found that expression of MEOX1 was highly enriched in tumor-infiltrating Tregs, and demonstrated that MEOX1 overexpression is sufficient to reprogram circulating Tregs to acquire the transcriptional and epigenetic landscape of tumor-infiltrating Tregs. Accordingly, enrichment of the MEOX1-dependent gene program in Tregs was strongly associated with poor prognosis in a large cohort of patients with iCCA. CONCLUSIONS: We observed abundant infiltration of hyperactivated CD4+ Tregs in iCCA tumors along with reduced CD8+ T-cell effector functions. Interfering with hyperactivated Tregs should be explored as an approach to enhance antitumor immunity in iCCA. LAY SUMMARY: Immune cells have the potential to slow or halt the progression of tumors. However, some tumors, such as intrahepatic cholangiocarcinoma, are associated with very limited immune responses (and infiltration of cancer-targeting immune cells). Herein, we show that a specific population of regulatory T cells (a type of immune cell that actually suppresses the immune response) are hyperactivated in intrahepatic cholangiocarcinoma. Targeting these cells could enable cancer-targeting immune cells to act more effectively and should be looked at as a potential therapeutic approach to this aggressive cancer type.

Macrophage expression and prognostic significance of the long pentraxin PTX3 in COVID-19.

Long pentraxin 3 (PTX3) is an essential component of humoral innate immunity, involved in resistance to selected pathogens and in the regulation of inflammation1-3. The present study was designed to assess the presence and significance of PTX3 in Coronavirus Disease 2019 (COVID-19)4-7. RNA-sequencing analysis of peripheral blood mononuclear cells, single-cell bioinformatics analysis and immunohistochemistry of lung autopsy samples revealed that myelomonocytic cells and endothelial cells express high levels of PTX3 in patients with COVID-19. Increased plasma concentrations of PTX3 were detected in 96 patients with COVID-19. PTX3 emerged as a strong independent predictor of 28-d mortality in multivariable analysis, better than conventional markers of inflammation, in hospitalized patients with COVID-19. The prognostic significance of PTX3 abundance for mortality was confirmed in a second independent cohort (54 patients). Thus, circulating and lung myelomonocytic cells and endothelial cells are a major source of PTX3, and PTX3 plasma concentration can serve as an independent strong prognostic indicator of short-term mortality in COVID-19.

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.

IRF4 instructs effector Treg differentiation and immune suppression in human cancer.

The molecular mechanisms responsible for the high immunosuppressive capacity of CD4+ Tregs in tumors are not well known. High-dimensional single-cell profiling of T cells from chemotherapy-naive individuals with non-small-cell lung cancer identified the transcription factor IRF4 as specifically expressed by a subset of intratumoral CD4+ effector Tregs with superior suppressive activity. In contrast to the IRF4- counterparts, IRF4+ Tregs expressed a vast array of suppressive molecules, and their presence correlated with multiple exhausted subpopulations of T cells. Integration of transcriptomic and epigenomic data revealed that IRF4, either alone or in combination with its partner BATF, directly controlled a molecular program responsible for immunosuppression in tumors. Accordingly, deletion of Irf4 exclusively in Tregs resulted in delayed tumor growth in mice while the abundance of IRF4+ Tregs correlated with poor prognosis in patients with multiple human cancers. Thus, a common mechanism underlies immunosuppression in the tumor microenvironment irrespective of the tumor type.

Immunological history governs human stem cell memory CD4 heterogeneity via the Wnt signaling pathway.

The diversity of the naïve T cell repertoire drives the replenishment potential and capacity of memory T cells to respond to immune challenges. Attrition of the immune system is associated with an increased prevalence of pathologies in aged individuals, but whether stem cell memory T lymphocytes (TSCM) contribute to such attrition is still unclear. Using single cells RNA sequencing and high-dimensional flow cytometry, we demonstrate that TSCM heterogeneity results from differential engagement of Wnt signaling. In humans, aging is associated with the coupled loss of Wnt/ß-catenin signature in CD4 TSCM and systemic increase in the levels of Dickkopf-related protein 1, a natural inhibitor of the Wnt/ß-catenin pathway. Functional assays support recent thymic emigrants as the precursors of CD4 TSCM. Our data thus hint that reversing TSCM defects by metabolic targeting of the Wnt/ß-catenin pathway may be a viable approach to restore and preserve immune homeostasis in the context of immunological history.

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.

Antioxidant metabolism regulates CD8+ T memory stem cell formation and antitumor immunity.

Adoptive T cell transfer (ACT) immunotherapy benefits from early differentiated stem cell memory T (Tscm) cells capable of persisting in the long term and generating potent antitumor effectors. Due to their paucity ex vivo, Tscm cells can be derived from naive precursors, but the molecular signals at the basis of Tscm cell generation are ill-defined. We found that less differentiated human circulating CD8+ T cells display substantial antioxidant capacity ex vivo compared with more differentiated central and effector memory T cells. Limiting ROS metabolism with antioxidants during naive T cell activation hindered terminal differentiation, while allowing expansion and generation of Tscm cells. N-acetylcysteine (NAC), the most effective molecule in this regard, induced transcriptional and metabolic programs characteristic of self-renewing memory T cells. Upon ACT, NAC-generated Tscm cells established long-term memory in vivo and exerted more potent antitumor immunity in a xenogeneic model when redirected with CD19-specific CAR, highlighting the translational relevance of NAC as a simple and inexpensive method to improve ACT.

Background fluorescence and spreading error are major contributors of variability in high-dimensional flow cytometry data visualization by t-distributed stochastic neighboring embedding.

Multidimensional single-cell analysis requires approaches to visualize complex data in intuitive 2D graphs. In this regard, t-distributed stochastic neighboring embedding (tSNE) is the most popular algorithm for single-cell RNA sequencing and cytometry by time-of-flight (CyTOF), but its application to polychromatic flow cytometry, including the recently developed 30-parameter platform, is still under investigation. We identified differential distribution of background values between samples, generated by either background calculation or spreading error (SE), as a major source of variability in polychromatic flow cytometry data representation by tSNE, ultimately resulting in the identification of erroneous heterogeneity among cell populations. Biexponential transformation of raw data and limiting SE during panel development dramatically improved data visualization. These aspects must be taken into consideration when using computational approaches as discovery tools in large sets of samples from independent experiments or immunomonitoring in clinical trials.

The early expansion of anergic NKG2Apos/CD56dim/CD16neg natural killer represents a therapeutic target in haploidentical hematopoietic stem cell transplantation.

Natural killer cells are the first lymphocyte population to reconstitute early after non-myeloablative and T cell-replete haploidentical hematopoietic stem cell transplantation with post-transplant infusion of cyclophosphamide. The study herein characterizes the transient and predominant expansion starting from the second week following haploidentical hematopoietic stem cell transplantation of a donor-derived unconventional subset of NKp46neg-low/CD56dim/CD16neg natural killer cells expressing remarkably high levels of CD94/NKG2A. Both transcription and phenotypic profiles indicated that unconventional NKp46neg-low/CD56dim/CD16neg cells are a distinct natural killer cell subpopulation with features of late stage differentiation, yet retaining proliferative capability and functional plasticity to generate conventional NKp46pos/CD56bright/CD16neg-low cells in response to interleukin-15 plus interleukin-18. While present at low frequency in healthy donors, unconventional NKp46neg-low/CD56dim/CD16neg cells are greatly expanded in the seven weeks following haploidentical hematopoietic stem cell transplantation, and express high levels of the activating receptors NKG2D and NKp30 as well as of the lytic granules Granzyme-B and Perforin. Nonetheless, NKp46neg-low/CD56dim/CD16neg cells displayed a markedly defective cytotoxicity that could be reversed by blocking the inhibitory receptor CD94/NKG2A. These data open new and important perspectives to better understand the ontogenesis/homeostasis of human natural killer cells and to develop a novel immune-therapeutic approach that targets the inhibitory NKG2A check-point, thus unleashing natural killer cell alloreactivity early after haploidentical hematopoietic stem cell transplantation.

Curtailed T-cell activation curbs effector differentiation and generates CD8+ T cells with a naturally-occurring memory stem cell phenotype.

Human T memory stem (TSCM ) cells with superior persistence capacity and effector functions are emerging as important players in the maintenance of long-lived T-cell memory and are thus considered an attractive population to be used in adoptive transfer-based immunotherapy of cancer. However, the molecular signals regulating their generation remain poorly defined. Here we show that curtailed T-cell receptor stimulation curbs human effector CD8+ T-cell differentiation and allows the generation of CD45RO- CD45RA+ CCR7+ CD27+ CD95+ -phenotype cells from highly purified naïve T-cell precursors, resembling naturally-occurring human TSCM . These cells proliferate extensively in vitro and in vivo, express low amounts of effector-associated genes and transcription factors and undergo considerable self-renewal in response to IL-15 while retaining effector differentiation potential. Such a phenotype is associated with a lower number of mitochondria compared to highly-activated effector T cells committed to terminal differentiation. These results shed light on the molecular signals that are required to generate long-lived memory T cells with potential application in adoptive cell transfer immunotherapy.

FACS Analysis of Memory T Lymphocytes.

Flow cytometry is a powerful and robust technology for detecting and monitoring multiple markers at the level of single cells. Since its early development, flow cytometry has been used to assess heterogeneity in a cell suspension. Over the years, the increasing number of parameters that could be included in a single assay combined with physical separation by fluorescence-activated cell sorting (FACS) revealed that the T cell compartment is extremely heterogenous in terms of phenotypic diversity, functional capacity, and transcriptional regulation. While naïve T cells are fairly homogenous, diversity becomes extreme in the antigen-experienced memory compartment. The precise identification of memory subsets by the simultaneous analysis of multiple markers by flow cytometry is key not only to basic science but also for the correct immunomonitoring of patients undergoing immunotherapy or for T cell-based therapeutic approaches. In this chapter, we provide guidelines to optimize complex flow cytometry panels, to achieve correct fluorescence compensation and determine positivity for a given antigen. Correct selection of reagents and their validation is essential to the success of the assay. Despite having been developed for the identification of human naïve and memory T cell subsets, the concepts illustrated here can be applied to any experiment aiming to investigate n parameters by flow cytometry.

Differentiation of Diverse Progenies of Memory T Cells from Naïve CD8+ T Cell Precursors.

Following recognition of the cognate antigen, naïve T cells differentiate in a diverse progeny of memory T cells which differ at the phenotypic, gene expression and metabolic level. These molecular differences are at the basis of discrete functionality, migratory capacity and persistence in the long-term. Such a division of labor benefits adoptive T cell transfer immunotherapy approaches for cancer and viral infections, as increased persistence and effector functions in vivo result in better control of the disease. Preclinical data suggest that early-differentiated T memory stem cells are the most powerful anti-tumor T cell population following adoptive transfer, but their paucity ex vivo limits translation to the clinic. Here, we describe a simple protocol to derive large numbers of T memory stem cell and effector CD8+ T cell subsets from highly-purified CD8+ naïve T cell precursors. The obtained cells can be studied in vitro to understand the molecular basis of human memory T cell differentiation, or, when redirected with T cell receptor or chimeric antigen receptor, potentially used in vivo to favour T cell reconstitution or to treat established tumors or chronic infections.

Role of naive-derived T memory stem cells in T-cell reconstitution following allogeneic transplantation.

Early T-cell reconstitution following allogeneic transplantation depends on the persistence and function of T cells that are adoptively transferred with the graft. Posttransplant cyclophosphamide (pt-Cy) effectively prevents alloreactive responses from unmanipulated grafts, but its effect on subsequent immune reconstitution remains undetermined. Here, we show that T memory stem cells (TSCM), which demonstrated superior reconstitution capacity in preclinical models, are the most abundant circulating T-cell population in the early days following haploidentical transplantation combined with pt-Cy and precede the expansion of effector cells. Transferred naive, but not TSCM or conventional memory cells preferentially survive cyclophosphamide, thus suggesting that posttransplant TSCM originate from naive precursors. Moreover, donor naive T cells specific for exogenous and self/tumor antigens persist in the host and contribute to peripheral reconstitution by differentiating into effectors. Similarly, pathogen-specific memory T cells generate detectable recall responses, but only in the presence of the cognate antigen. We thus define the cellular basis of T-cell reconstitution following pt-Cy at the antigen-specific level and propose to explore naive-derived TSCM in the clinical setting to overcome immunodeficiency. These trials were registered at www.clinicaltrials.gov as #NCT02049424 and #NCT02049580.