A Cas9 nanoparticle system with truncated Cas9 target sequences on DNA repair templates enhances genome targeting in diverse human immune cell types

ABSTRACTVirus-modified T cells are approved for cancer immunotherapy, but more versatile and precise genome modifications are needed for a wider range of adoptive cellular therapies1–4. We recently developed a non-viral CRISPR–Cas9 system for genomic site-specific integration of large DNA sequences in primary human T cells5. Here, we report two key improvements for efficiency and viability in an expanded variety of clinically-relevant primary cell types. We discovered that addition of truncated Cas9 target sequences (tCTS) at the ends of the homology directed repair (HDR) templates can interact with Cas9 ribonucleoproteins (RNPs) to ‘shuttle’ the template and enhance targeting efficiency. Further, stabilizing the Cas9 RNPs into nanoparticles with poly(glutamic acid) improved editing, reduced toxicity, and enabled lyophilized storage without loss of activity. Combining the tCTS HDR template modifications with polymer-stabilized nanoparticles increased gene targeting efficiency and viable cell yield across multiple genomic loci in diverse cell types. This system is an inexpensive, user-friendly delivery platform for non-viral genome reprogramming that we successfully applied in regulatory T cells (Tregs), γδ-T cells, B cells, NK cells, and primary and iPS-derived6 hematopoietic stem progenitor cells (HSPCs).

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Epidermal γδ T cells originate from yolk sac hematopoiesis and clonally self-renew in the adult

Journal of Experimental Medicine ◽

10.1084/jem.20181206 ◽

2018 ◽

Vol 215 (12) ◽

pp. 2994-3005 ◽

Cited By ~ 25

Author(s):

Rebecca Gentek ◽

Clément Ghigo ◽

Guillaume Hoeffel ◽

Audrey Jorquera ◽

Rasha Msallam ◽

...

Keyword(s):

T Cells ◽

Immune Cell ◽

Fetal Liver ◽

Γδ T Cells ◽

Yolk Sac ◽

Lineage Tracing ◽

Hematopoietic Stem ◽

Turn Over ◽

Initial Seeding ◽

Mapping Systems

The murine epidermis harbors two immune cell lineages, Langerhans cells (LCs) and γδ T cells known as dendritic epidermal T cells (DETCs). LCs develop from both early yolk sac (YS) progenitors and fetal liver monocytes before locally self-renewing in the adult. For DETCs, the mechanisms of homeostatic maintenance and their hematopoietic origin are largely unknown. Here, we exploited multicolor fate mapping systems to reveal that DETCs slowly turn over at steady state. Like for LCs, homeostatic maintenance of DETCs is achieved by clonal expansion of tissue-resident cells assembled in proliferative units. The same mechanism, albeit accelerated, facilitates DETC replenishment upon injury. Hematopoietic lineage tracing uncovered that DETCs are established independently of definitive hematopoietic stem cells and instead originate from YS hematopoiesis, again reminiscent of LCs. DETCs thus resemble LCs concerning their maintenance, replenishment mechanisms, and hematopoietic development, suggesting that the epidermal microenvironment exerts a lineage-independent influence on the initial seeding and homeostatic maintenance of its resident immune cells.

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The Aging of γδ T Cells

Cells ◽

10.3390/cells9051181 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1181 ◽

Cited By ~ 5

Author(s):

Weili Xu ◽

Zandrea Wan Xuan Lau ◽

Tamas Fulop ◽

Anis Larbi

Keyword(s):

T Cells ◽

Immune System ◽

Immune Cell ◽

Γδ T Cells ◽

Cell Types ◽

Developed Countries ◽

Common Denominator ◽

Age Related ◽

The Relationship ◽

Diverse Interactions

In the coming decades, many developed countries in the world are expecting the “greying” of their populations. This phenomenon poses unprecedented challenges to healthcare systems. Aging is one of the most important risk factors for infections and a myriad of diseases such as cancer, cardiovascular and neurodegenerative diseases. A common denominator that is implicated in these diseases is the immune system. The immune system consists of the innate and adaptive arms that complement each other to provide the host with a holistic defense system. While the diverse interactions between multiple arms of the immune system are necessary for its function, this complexity is amplified in the aging immune system as each immune cell type is affected differently—resulting in a conundrum that is especially difficult to target. Furthermore, certain cell types, such as γδ T cells, do not fit categorically into the arms of innate or adaptive immunity. In this review, we will first introduce the human γδ T cell family and its ligands before discussing parallels in mice. By covering the ontogeny and homeostasis of γδ T cells during their lifespan, we will better capture their evolution and responses to age-related stressors. Finally, we will identify knowledge gaps within these topics that can advance our understanding of the relationship between γδ T cells and aging, as well as age-related diseases such as cancer.

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Implications of hematopoietic stem cells heterogeneity for gene therapies

Gene Therapy ◽

10.1038/s41434-021-00229-x ◽

2021 ◽

Author(s):

Jeremy Epah ◽

Richard Schäfer

Keyword(s):

Immune Cell ◽

Ex Vivo ◽

Bone Marrow Failure ◽

Cell Types ◽

Blood Disorders ◽

Hematopoietic Stem ◽

Therapeutic Concept ◽

Gene Therapies ◽

Bone Marrow Failure Syndromes ◽

Immunodeficiency Syndrome

AbstractHematopoietic stem cell transplantation (HSCT) is the therapeutic concept to cure the blood/immune system of patients suffering from malignancies, immunodeficiencies, red blood cell disorders, and inherited bone marrow failure syndromes. Yet, allogeneic HSCT bear considerable risks for the patient such as non-engraftment, or graft-versus host disease. Transplanting gene modified autologous HSCs is a promising approach not only for inherited blood/immune cell diseases, but also for the acquired immunodeficiency syndrome. However, there is emerging evidence for substantial heterogeneity of HSCs in situ as well as ex vivo that is also observed after HSCT. Thus, HSC gene modification concepts are suggested to consider that different blood disorders affect specific hematopoietic cell types. We will discuss the relevance of HSC heterogeneity for the development and manufacture of gene therapies and in exemplary diseases with a specific emphasis on the key target HSC types myeloid-biased, lymphoid-biased, and balanced HSCs.

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Diversity, localization and (patho)physiology of mature lymphocyte populations in the bone marrow

Blood ◽

10.1182/blood.2020007592 ◽

2021 ◽

Author(s):

Christian M. Schürch ◽

Chiara Caraccio ◽

Martijn A. Nolte

Keyword(s):

Bone Marrow ◽

T Cells ◽

Plasma Cells ◽

Cell Types ◽

Lymphoid Organ ◽

Lymphoid Cells ◽

Term Survival ◽

Hematopoietic Stem ◽

Mature Lymphocyte ◽

Lymphocyte Populations

The bone marrow (BM) is responsible for generating and maintaining lifelong output of blood and immune cells. Besides its key hematopoietic function, the BM acts as an important lymphoid organ, hosting a large variety of mature lymphocyte populations, including B-cells, T-cells, NK(T)-cells and innate lymphoid cells (ILCs). Many of these cell types are thought to only transiently visit the BM, but for others, like plasma cells and memory T-cells, the BM provides supportive niches that promote their long-term survival. Interestingly, accumulating evidence points towards an important role for mature lymphocytes in the regulation of hematopoietic stem cells (HSCs) and hematopoiesis in health and disease. In this review, we describe the diversity, migration, localization and function of mature lymphocyte populations in murine and human BM, focusing on their role in immunity and hematopoiesis. We also address how various BM lymphocyte subsets contribute to the development of aplastic anemia and immune thrombocytopenia, illustrating the complexity of these BM disorders, but also the underlying similarities and differences in their disease pathophysiology. Finally, we summarize the interactions between mature lymphocytes and BM resident cells in HSC transplantation and graft-versus-host disease. A better understanding of the mechanisms by which mature lymphocyte populations regulate BM function will likely improve future therapies for patients with benign and malignant hematological disorders.

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Peripheral Immunological Cells in Pregnant Women and their Change during Diabetes

Experimental and Clinical Endocrinology & Diabetes ◽

10.1055/s-0043-104935 ◽

2017 ◽

Vol 125 (10) ◽

pp. 677-683 ◽

Cited By ~ 1

Author(s):

Ulrike Friebe-Hoffmann ◽

Linda Antony ◽

Jan-Steffen Kruessel ◽

Brigitte Pawlowski ◽

Thomas Hoffmann

Keyword(s):

T Cells ◽

Gestational Diabetes ◽

Pregnant Women ◽

Immune Cell ◽

Γδ T Cells ◽

Complication Rates ◽

And Shifts ◽

The Impact ◽

Pregnant Diabetic

AbstractDuring the last decades the incidence of diabetes has dramatically increased as well as the number of pregnant diabetic women. There is still missing data regarding patterns and shifts of immune cell populations due to pregnancy with or without diabetes. The study aimed to investigate the impact of pregnancy, type 1 diabetes (T1D) and gestational diabetes mellitus (GDM) on different immune cells in female. The number and proportion of CD3-, CD4-, CD8- and γδ T-cells as well as B-, NK-, NKT- and dendritic cells (DC) incl. rate of apoptosis was analyzed in peripheral blood samples from 24 non-pregnant women, 24 pregnant controls, 25 non-pregnant T1D, 18 women with GDM and 15 pregnant T1D (PT1D) women. Compared to healthy controls, healthy pregnant women had reduced numbers of lymphoid DC and γδ T-cells, while women with gestational diabetes presented with increased numbers of γδ T-cells. Pregnant women with T1D showed increased NKT cells and a decrease of NK cells compared to healthy pregnant or non-pregnant T1D women. Apoptosis of γδ T-cells in healthy pregnant women was found to be decreased in comparison to their non-pregnant controls while apoptosis of myeloid and lymphoid DC was increased in pregnant T1D in comparison to non-pregnant T1D. Those results may indicate that increased complication rates during diabetic pregnancies might be due to an impaired adaptation of the immune system.

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A new family of markers to characterize the human γδ T-cell lineage

10.31219/osf.io/jzcf3 ◽

2019 ◽

Author(s):

Shahan Mamoor

Keyword(s):

T Cells ◽

T Cell ◽

Differentially Expressed Genes ◽

Cell Lineage ◽

Γδ T Cells ◽

Differentially Expressed ◽

Γδ T Cell ◽

Differentiation Markers ◽

Hematopoietic Stem ◽

New Family

Prospective isolation of γδ T lymphocytes demands a comprehensive description of the molecules that distinguish T cells with γδ T-cell receptors (TCRs) (γδ T cells, or Tγδ) from those with αβTCRs (Tαβ). Here I describe some of the most differentially expressed genes in the γδ T cell when compared to the developmentally proximal but lineage-distinct Tαβ CD4+ and CD8+ lymphocytes. These genes encode cluster of differentiation markers, transcription factors, cell surface receptors and non-coding RNAs. As hematopoietic stem cells (HSCs) have been prospectively isolated based on the analysis of differentially expressed genes (1), any combination of these molecules may potentially be used to isolate Tγδ, perhaps even independent of the γδTCR. This description of the most striking identifying features of the Tγδ will be a resource for the isolation of a multi-potent common γδ T-cell progenitor.

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GSDMA drives the most replicated association with asthma in naïve CD4+ T cells

10.1101/774760 ◽

2019 ◽

Cited By ~ 1

Author(s):

Anne-Marie Madore ◽

Lucile Pain ◽

Anne-Marie Boucher-Lafleur ◽

Jolyane Meloche ◽

Andréanne Morin ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

T Cells ◽

Genetic Variants ◽

Immune Cell ◽

Cell Types ◽

Sequencing Data ◽

Opposite Pattern ◽

Genetic Mechanisms ◽

New Gene

AbstractBackgroundThe 17q12-21 locus is the most replicated association with asthma. However, no study had described the genetic mechanisms underlying this association considering all genes of the locus in immune cell samples isolated from asthmatic and non-asthmatic individuals.ObjectiveThis study takes benefit of samples from naïve CD4+ T cells and eosinophils isolated from the same 200 individuals to describe specific interactions between genetic variants, gene expression and DNA methylation levels for the 17q12-21 asthma locus.Methods and ResultsAfter isolation of naïve CD4+ T cells and eosinophils from blood samples, next generation sequencing was used to measure DNA methylation levels and gene expression counts. Genetic interactions were then evaluated considering genetic variants from imputed genotype data. In naïve CD4+ T cells but not eosinophils, 20 SNPs in the fourth and fifth haplotype blocks modulated both GSDMA expression and methylation levels, showing an opposite pattern of allele frequencies and expression counts in asthmatics compared to controls. Moreover, negative correlations have been measured between methylation levels of CpG sites located within the 1.5 kb region from the transcription start site of GSDMA and its expression counts.ConclusionAvailability of sequencing data from two key cell types isolated from asthmatic and non-asthmatic individuals allowed identifying a new gene in naïve CD4+ T cells that drives the association with the 17q12-21 locus, leading to a better understanding of the genetic mechanisms taking place in it.

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Extreme disruption of heterochromatin is required for accelerated hematopoietic aging

Blood ◽

10.1182/blood.2019002990 ◽

2020 ◽

Vol 135 (23) ◽

pp. 2049-2058 ◽

Cited By ~ 4

Author(s):

Christine R. Keenan ◽

Nadia Iannarella ◽

Gaetano Naselli ◽

Naiara G. Bediaga ◽

Timothy M. Johanson ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Cell Function ◽

Cell Types ◽

Premature Aging ◽

Thymic Involution ◽

Hematopoietic Stem ◽

Hematopoietic System

Abstract Loss of heterochromatin has been proposed as a universal mechanism of aging across different species and cell types. However, a comprehensive analysis of hematopoietic changes caused by heterochromatin loss is lacking. Moreover, there is conflict in the literature around the role of the major heterochromatic histone methyltransferase Suv39h1 in the aging process. Here, we use individual and dual deletion of Suv39h1 and Suv39h2 enzymes to examine the causal role of heterochromatin loss in hematopoietic cell development. Loss of neither Suv39h1 nor Suv39h2 individually had any effect on hematopoietic stem cell function or the development of mature lymphoid or myeloid lineages. However, deletion of both enzymes resulted in characteristic changes associated with aging such as reduced hematopoietic stem cell function, thymic involution and decreased lymphoid output with a skewing toward myeloid development, and increased memory T cells at the expense of naive T cells. These cellular changes were accompanied by molecular changes consistent with aging, including alterations in nuclear shape and increased nucleolar size. Together, our results indicate that the hematopoietic system has a remarkable tolerance for major disruptions in chromatin structure and reveal a role for Suv39h2 in depositing sufficient H3K9me3 to protect the entire hematopoietic system from changes associated with premature aging.

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High-dimensional cytometric analysis of colorectal cancer reveals novel mediators of antitumour immunity

Gut ◽

10.1136/gutjnl-2019-318672 ◽

2019 ◽

Vol 69 (4) ◽

pp. 691-703 ◽

Cited By ~ 21

Author(s):

Natasja L de Vries ◽

Vincent van Unen ◽

Marieke E Ijsselsteijn ◽

Tamim Abdelaal ◽

Ruud van der Breggen ◽

...

Keyword(s):

Colorectal Cancer ◽

T Cells ◽

Lymph Nodes ◽

Single Cell ◽

Immune Cell ◽

Γδ T Cells ◽

High Dimensional ◽

Hla Dr ◽

Healthy Mucosa ◽

Immune Contexture

ObjectiveA comprehensive understanding of anticancer immune responses is paramount for the optimal application and development of cancer immunotherapies. We unravelled local and systemic immune profiles in patients with colorectal cancer (CRC) by high-dimensional analysis to provide an unbiased characterisation of the immune contexture of CRC.DesignThirty-six immune cell markers were simultaneously assessed at the single-cell level by mass cytometry in 35 CRC tissues, 26 tumour-associated lymph nodes, 17 colorectal healthy mucosa and 19 peripheral blood samples from 31 patients with CRC. Additionally, functional, transcriptional and spatial analyses of tumour-infiltrating lymphocytes were performed by flow cytometry, single-cell RNA-sequencing and multispectral immunofluorescence.ResultsWe discovered that a previously unappreciated innate lymphocyte population (Lin–CD7+CD127–CD56+CD45RO+) was enriched in CRC tissues and displayed cytotoxic activity. This subset demonstrated a tissue-resident (CD103+CD69+) phenotype and was most abundant in immunogenic mismatch repair (MMR)-deficient CRCs. Their presence in tumours was correlated with the infiltration of tumour-resident cytotoxic, helper and γδ T cells with highly similar activated (HLA-DR+CD38+PD-1+) phenotypes. Remarkably, activated γδ T cells were almost exclusively found in MMR-deficient cancers. Non-activated counterparts of tumour-resident cytotoxic and γδ T cells were present in CRC and healthy mucosa tissues, but not in lymph nodes, with the exception of tumour-positive lymph nodes.ConclusionThis work provides a blueprint for the understanding of the heterogeneous and intricate immune landscape of CRC, including the identification of previously unappreciated immune cell subsets. The concomitant presence of tumour-resident innate and adaptive immune cell populations suggests a multitargeted exploitation of their antitumour properties in a therapeutic setting.

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Analysis Of Immune Cell Populations Before and After Autologous Hematopoietic Stem Cell Transplant For Multiple Myeloma: Association With Early Recovery Of Absolute Lymphocyte Count and Progression-Free Survival

Blood ◽

10.1182/blood.v122.21.3348.3348 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3348-3348

Author(s):

Theresa Hahn ◽

Yali Zhang ◽

Bruno Paiva ◽

George L. Chen ◽

Paul K. Wallace ◽

...

Keyword(s):

T Cells ◽

Hematopoietic Stem Cell Transplant ◽

Immune Cell ◽

Absolute Lymphocyte Count ◽

Central Memory ◽

Cell Transplant ◽

Early Recovery ◽

Hematopoietic Stem ◽

Immune Cell Subsets ◽

Clinically Significant

Abstract The treatment of transplant-eligible, multiple myeloma (MM) patients usually consists of induction therapy followed by autologous hematopoietic stem cell transplant (ASCT). In addition to typical cytogenetic and biochemical prognostic factors, there are other risk factors that predict for relapse and progression after ASCT. This includes absolute lymphocyte count (ALC) recovery (>0.5 cells/μL) by day +15 post ASCT, which has previously been shown to correlate with prolonged progression-free (PFS) and overall survival (OS). In a cohort of 70 MM patients who received high dose melphalan with ASCT between 8/2007 and 9/2012, we performed a comprehensive immunophenotyping panel including 35 T-, B-, NK- and Dendritic Cell (DC)-cell subsets a median of 25 days before ASCT (N=70), at day +30 post-ASCT (N=40) and at day +100 (N=51) post-ASCT. Specifically, the immunophenotyping panel included total numbers of T cells (CD3+, CD4+, CD8+), B cells (CD19+, CD20+), NK cells (CD56+CD16+) and myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells. T cell subsets included the relative proportions of double positive (CD3+CD4+CD8+), double negative (CD3+CD4-CD8-), CD4+ and CD8+ Naïve (CD3+ CD45RA+ CD45RO- CD27+), CD4+ and CD8+ central memory (CD3+ CD45RA- CD45RO+ CD27+), CD4+ and CD8+ effector memory (CD3+ CD45RA- CD45RO+ CD27-), recent thymic emigrants (CD3+ CD4+ CD45RA+ CD31+), and T-regulatory cells (CD3+ CD4+ CD25(br), CD3+CD4+CD25+CD127(d), CD3+CD4+CD25+CD127-HLADr+). B cell subsets included proportions of naïve (CD19+ CD27-), and memory (CD19+ CD27+, pre-switch CD19+ IgD+ CD27+, post-switch CD19+ IgD- CD27) cells. All patients survived to day +100. We examined MM response to ASCT, recovery of ALC by day +15 post-ASCT, clinically significant infections, PFS and OS. Characteristics in 70 patients pre-ASCT were: median (range) age 60 (28-75) yrs; 54% females; 26% KPS ≥90; response to most recent therapy was 13% in CR/sCR, 40% in VGPR, 41% in PR, 6% stable disease. MM responses at day + 100 included: 51% achieved or maintained CR/sCR, 17% VGPR, 10% PR, 24% SD and 6% progressed. The frequency of different immune cell subsets at either pre-ASCT or day +100 post-ASCT were not associated with CR/sCR status at day 100 post-AHSCT. ALC recovery by day +15 post-ASCT was associated with significantly longer median PFS (651 vs 288 days, P=0.001) and OS (804 vs 391 days, P=0.02) than those who did not recover ALC by day 15. For patients showing early recovery of ALC, the absolute numbers of T-cells pre-ASCT (P=0.08), as well as the proportion of CD4+ naïve (P=0.07) and CD8+ NK (CD8+/CD16+/CD56+, P=0.06) populations tended to be higher, whereas the absolute counts of gamma-delta T cells (P=0.02), CD4+ helper (P=0.03, See Figure), CD8+ effector (P=0.02) T-cells and total B-cell count (P=0.05) were significantly higher as compared to patients who did not achieve ALC recovery by day +15. Of note, within the T-cell compartment, CD4+ central memory (P=0.02, see Figure), CD8+ central memory (P=0.05), T-regulatory (T reg) (P=0.02, CD4+25+) populations were significantly lower. The distribution of different immune cell subsets pre-ASCT was not associated with development of clinically significant infections before day +100 post-AHCT; however, the proportion of memory B cells measured at day+100 was significantly higher (p=0.02) in patients who had experienced an infection before day +100. In the present study we show that the distribution of different immune cell populations correlates with ALC recovery in MM. Moreover, we also confirmed the association of ALC recovery with prolonged PFS and OS. The increase in the relative proportions of CD4+ and CD8+ effector cell subpopulations and the decrease in CD4+ and CD8+ central memory and T-reg populations demonstrate that the pre-ASCT effector cell phenotype contributes to faster ALC recovery, and improved PFS/OS. Therapeutic strategies to alter the pre-ASCT and/or day + 100 immune phenotype may improve outcomes after ASCT.Figure 1Figure 1. Figure 2Figure 2. Disclosures: McCarthy: Janssen: Honoraria; Celgene: Consultancy.

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