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Imaging Mass Cytometry™ (IMC™) is a vital tool to deeply characterize the complexity and diversity of any tissue without disrupting spatial context. The Hyperion™ Imaging System utilizes IMC, based on CyTOF® technology, to simultaneously assess up to 40 individual structural and functional markers in tissues, providing unprecedented insight into the organization and function of tissue microenvironment. We have previously demonstrated the application of IMC in combination with Maxpar® panel kits to highlight cellular composition of human tissues. Here, we showcase the recently released Maxpar OnDemand Antibodies for IMC application on mouse tissue. We introduced 11 additional biomarkers to our existing mouse antibody catalog, providing the basis for the use of high-multiplex imaging in preclinical investigations. To demonstrate the IMC workflow on mouse tissue, we analyzed a normal mouse tissue microarray using IMC spatial proteomic analysis. Tissues were stained with a 20-marker panel designed to highlight tissue architecture and major immune lineage markers combined with our IMC Cell Segmentation Kit*. The IMC Cell Segmentation Kit facilitates identification of cellular borders using plasma membrane markers that lead to improved nucleus and plasma membrane demarcation. We generated a detailed spatial map of the heterogeneous tissue architecture and successfully identified immune, epithelial, and stromal cell populations in various mouse tissues. Additionally, we classified the activation state of immune cell populations, adhesion state of epithelial cells, and molecular composition of the extracellular matrix.Overall, this work demonstrates the capability of IMC to identify subcellular localization of cellular and structural markers in the mouse tissue microenvironment. Information gained from IMC studies will enable in-depth high-throughput phenotypic characterization of the tissue microenvironment in various mouse models of development and disease, and thus accelerate preclinical discoveries. *The IMC Cell Segmentation Kit is part of the Innovative Solutions menu of custom-made reagents and workflows developed and tested by Fluidigm scientists to give faster access to new cutting-edge solutions for high-multiplex single-cell analysis. Innovative Solutions are not part of the Maxpar catalog. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Qanber Raza, Michael Cohen, Smriti Kala, Liang Lim, Geneve Awong, Andrew Quong, Christina Loh. Imaging mass cytometry identifies structural and cellular composition of the mouse tissue microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2035.

Imaging Mass Cytometry™ (IMC™) is a vital tool to deeply characterize the complexity and diversity of any tissue without disrupting spatial context. The Hyperion™ Imaging System utilizes IMC, based on CyTOF® technology, to assess up to 40 individual structural and functional markers in tissues, providing unprecedented insight into the organization and function of tissue microenvironment. We have previously demonstrated the application of IMC in combination with Maxpar® panel kits on human tissues. Here, we showcase Maxpar OnDemand Antibodies for IMC application including 11 new highly relevant markers to construe cellular and molecular composition of mouse tissues. We analyzed a normal mouse tissue microarray using IMC spatial proteomic analysis. Tissues were stained with a 20-marker panel designed to highlight tissue architecture and major immune lineage markers. We generated a detailed spatial map of the diverse tissue architecture and successfully identified immune, epithelial, and stromal cell populations in various mouse tissues. Additionally, we classified the activation state of immune cell populations, adhesion state of epithelial cells, and molecular composition of the extracellular matrix. This work demonstrates the capability of IMC to identify subcellular localization of cellular and structural markers in the mouse tissue microenvironment. Future studies utilizing IMC in combination with Maxpar OnDemand Antibodies will enable in-depth phenotypic characterization of the tissue microenvironment in various mouse models of development and disease, and thus provide the basis for the use of high-multiplex imaging in preclinical investigations. For Research Use Only. Not for use in diagnostic procedures.

SARS-CoV-2 infection can affect all human beings, including pregnant women. Thus, understanding the immunological changes induced by the virus during pregnancy is nowadays of pivotal importance. Here, using peripheral blood from 14 pregnant women with asymptomatic or mild SARS-CoV-2 infection, we investigate cell proliferation and cytokine production, measure plasma levels of 62 cytokines, and perform a 38-parameter mass cytometry analysis. Our results show an increase in low density neutrophils but no lymphopenia or gross alterations of white blood cells, which display normal levels of differentiation, activation or exhaustion markers and show well preserved functionality. Meanwhile, the plasma levels of anti-inflammatory cytokines such as interleukin (IL)-1RA, IL-10 and IL-19 are increased, those of IL-17, PD-L1 and D-dimer are decreased, but IL-6 and other inflammatory molecules remain unchanged. Our profiling of antiviral immune responses may thus help develop therapeutic strategies to avoid virus-induced damages during pregnancy.

The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) will depend on the accurate recapitulation of embryonic haematopoiesis. In the early embryo, HSCs develop from the haemogenic endothelium (HE) and are specified in a Notch-dependent manner through a process named endothelial-to-haematopoietic transition (EHT). As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE). Here we demonstrate at a clonal level that hPSC-derived HE and VE represent separate lineages. HE is restricted to the CD34(+)CD73(-)CD184(-) fraction of day 8 embryoid bodies and it undergoes a NOTCH-dependent EHT to generate RUNX1C(+) cells with multilineage potential. Arterial and venous VE progenitors, in contrast, segregate to the CD34(+)CD73(med)CD184(+) and CD34(+)CD73(hi)CD184(-) fractions, respectively. Together, these findings identify HE as distinct from VE and provide a platform for defining the signalling pathways that regulate their specification to functional HSCs.

SummaryThe efficient generation of hematopoietic stem cells from human pluripotent stem cells is dependent on the appropriate specification of the definitive hematopoietic program during differentiation. In this study, we used T lymphocyte potential to track the onset of definitive hematopoiesis from human embryonic and induced pluripotent stem cells differentiated with specific morphogens in serum- and stromal-free cultures. We show that this program develops from a progenitor population with characteristics of hemogenic endothelium, including the expression of CD34, VE-cadherin, GATA2, LMO2, and RUNX1. Along with T cells, these progenitors display the capacity to generate myeloid and erythroid cells. Manipulation of Activin/Nodal signaling during early stages of differentiation revealed that development of the definitive hematopoietic progenitor population is not dependent on this pathway, distinguishing it from primitive hematopoiesis. Collectively, these findings demonstrate that it is possible to generate T lymphoid progenitors from pluripotent stem cells and that this lineage develops from a population whose emergence marks the onset of human definitive hematopoiesis.

The thymus provides a unique and essential microenvironment for T-cell precursors to develop into mature functionally competent T lymphocytes. Ageing causes architectural changes in the thymus resulting in a loss of thymic epithelial space required for thymopoiesis - a process known as thymic involution. Additionally, cytoablative regimens used to treat malignancies also destroy thymic architecture. The net result of both processes is diminished thymic output and function that may lead to impaired immunity. Thus, immunocompromised individuals would benefit from strategies aimed at enhancing T-cell reconstitution.Here we discuss strategies such as the use of sex steroid ablation, keratinocyte growth factor, interleukin-7, and in-vitro-generated progenitor T cells as candidates for restoring T-cell immunity. Using various animal models of ageing or hematopoietic stem cell transplantation, these strategies have been shown to restore thymic architecture and cellularity, resulting in increased output and T-cell function in the periphery.These candidate approaches are currently being tested in clinical trials, with preliminary evidence showing encouraging effects on T-cell reconstitution. Nevertheless, although these strategies show clear promise in animal models, and in early human trials, further data are needed to determine their efficacy in patients.

T-cell development follows a defined set of stage-specific differentiation steps. However, molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. To address this, human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T lineage in OP9-DL1 cocultures. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. Quantitative clonal analyses demonstrated that CD34(+)CD38(-) and CD34(+)CD38(lo) subsets of UCB contain a similarly high T-lineage progenitor frequency, whereas the frequency in CD34(+)CD38(+/hi) cells was 5-fold lower. Delta-like/Notch-induced signals increased the T-cell progenitor frequency of CD34(+)CD38(-/lo) cells differentiated on OP9-DL1, and 2 distinct progenitor subsets, CD34(+)CD45RA(+)CD7(++)CD5(-)CD1a(-) (proT1) and CD34(+)CD45RA(+)CD7(++)CD5(+)CD1a(-) (proT2), were identified and their thymus engrafting capacity was examined, with proT2 cells showing a 3-fold enhanced reconstituting capacity compared with the proT1 subset. Furthermore, in vitro-generated CD34(+)CD7(++) progenitors effectively engrafted the thymus of immunodeficient mice, which was enhanced by the addition of an IL-7/IL-7 antibody complex. Taken together, the identification of T-progenitor subsets readily generated in vitro may offer important avenues to improve cellular-based immune-reconstitution approaches.

Primary immunodeficiency diseases comprise a group of heterogeneous genetic defects that affect immune system development and/or function. Here we use in vitro differentiation of human induced pluripotent stem cells (iPSCs) generated from patients with different recombination-activating gene 1 (RAG1) mutations to assess T-cell development and T-cell receptor (TCR) V(D)J recombination. RAG1-mutants from severe combined immunodeficient (SCID) patient cells showed a failure to sustain progression beyond the CD3(--)CD4(-)CD8(-)CD7(+)CD5(+)CD38(-)CD31(-/lo)CD45RA(+) stage of T-cell development to reach the CD3(-/+)CD4(+)CD8(+)CD7(+)CD5(+)CD38(+)CD31(+)CD45RA(-) stage. Despite residual mutant RAG1 recombination activity from an Omenn syndrome (OS) patient, similar impaired T-cell differentiation was observed, due to increased single-strand DNA breaks that likely occur due to heterodimers consisting of both an N-terminal truncated and a catalytically dead RAG1. Furthermore, deep-sequencing analysis of TCR-β (TRB) and TCR-α (TRA) rearrangements of CD3(-)CD4(+)CD8(-) immature single-positive and CD3(+)CD4(+)CD8(+) double-positive cells showed severe restriction of repertoire diversity with preferential usage of few Variable, Diversity, and Joining genes, and skewed length distribution of the TRB and TRA complementary determining region 3 sequences from SCID and OS iPSC-derived cells, whereas control iPSCs yielded T-cell progenitors with a broadly diversified repertoire. Finally, no TRA/δ excision circles (TRECs), a marker of TRA/δ locus rearrangements, were detected in SCID and OS-derived T-lineage cells, consistent with a pre-TCR block in T-cell development. This study compares human T-cell development of SCID vs OS patients, and elucidates important differences that help to explain the wide range of immunologic phenotypes that result from different mutations within the same gene of various patients.

Interleukin-4 (IL-4) is a multifunctional cytokine, which is involved in numerous disease states, including atopic asthma. IL-4 not only induces direct responses in cells but can also prime for secondary responses to stimuli. Little is known about the priming effects of IL-4 on endothelial cells; therefore, we chose to examine the ability of IL-4 to prime endothelial cells for platelet-activating factor (PAF) synthesis and prostaglandin E2 (PGE2) release. IL-4 alone did not enhance PAF synthesis or PGE2 release; however, pretreatment with IL-4 primed for PAF synthesis and PGE2 release in response to subsequent stimulation with histamine. In contrast, tumor necrosis factor α (TNF-α), oncostatin M (OSM), and IL-1β did not prime endothelial cells for PAF synthesis in response to histamine. The priming effects of IL-4 occurred without any detectable changes in the requirement for signaling pathways upstream of PGE2 release. IL-4 treatment increased the expression of mRNA for histamine receptor 1 (HR1) and shifted the inhibition curve for pyrilamine, a specific HR1 antagonist. In addition, the dose-response curve for histamine-induced elevations in intracellular calcium was shifted following IL-4 stimulation. Together, these data indicate that HR1 is up-regulated in IL-4-stimulated human umbilical vein endothelial cells (HUVEC) and suggest that this up-regulation may contribute to the enhanced responsiveness of IL-4-stimulated HUVEC to histamine.

Innate lymphoid cells (ILC) are RAG-independent lymphocytes with important roles in innate immunity, and include group-1 (natural killer (NK) cell, ILC1), group-2 (ILC2), and group-3 (lymphoid tissue inducer (LTi), NCR(+) ILC3) subsets. Group-3 ILC express Rorγt, produce interleukin (IL)-22, and are critically important in the normal function of mucosal tissues. Here, we describe a novel model cell line for the study of ILC function and differentiation. The parental MNK cell line, derived from NKR-P1B(+) fetal thymocytes, shows a capacity to differentiate in γc cytokines. One IL-7-responsive subline, designated MNK-3, expresses Rorγt and produces high levels of IL-22 in response to IL-23 and IL-1β stimulation. MNK-3 cells display surface markers and transcript expression characteristic of group-3 ILC, including IL-7Rα (CD127), c-kit (CD117), CCR6, Thy1 (CD90), RANK, RANKL, and lymphotoxin (LTα1β2). Using an in vitro assay of LTi cell activity, MNK-3 cells induce ICAM-1 and VCAM-1 expression on stromal cells in a manner dependent upon LTα1β2 expression. A second IL-2-responsive subline, MNK-1, expresses several NK cell receptors, perforin and granzymes, and shows some cytotoxic activity. Thus, MNK-1 cells serve as a model of ILC1/NK development and differentiation, whereas MNK-3 cells provide an attractive in vitro system to study the function of ILC3/LTi cells.

The thymus provides a unique environment for the induction of T-cell lineage commitment and differentiation, which is predicted by specific Notch ligand-receptor interactions on epithelial cells and lymphoid progenitors, respectively. Accordingly, a bone marrow-derived stromal cell line (OP9) ectopically expressing the Notch ligand Delta-like 1 (Dll1) or Dll4 (OP9-DL1 and OP9-DL4, respectively) gains the ability to recapitulate thymus-like function, supporting T-cell differentiation of both mouse and human progenitors. In this study, we extend these findings by demonstrating that, unlike the NIH3T3 cell line, mouse primary fibroblasts made to express Dll4 (mFibro-DL4) acquire the capacity to promote and support T-cell development from hematopoietic stem cells (HSCs) into TCRαβ(+), CD4(+) and CD8(+) T-lineage cells. However, mFibro-DL4 cells showed a lower efficiency of T-cell generation than OP9-DL4 cells did. Nevertheless, progenitor T-cells (CD117(+) CD44(+) CD25(+)) generated in HSC/mFibro-DL4 co-cultures, when intravenously transferred into immunodeficient (Rag2(-/-) γc(-/-)) mice, home to the thymus, undergo differentiation, and give rise to mature T-cells that go on to populate the periphery. Surprisingly, primary human fibroblast cells expressing Dll4 showed very low efficiency in supporting human T-lineage differentiation, which could not be improved by blocking myelopoiesis. Nevertheless, mFibro-DL4 cells could support human T-cell lineage differentiation. Our results provide a functional framework for the induction of T-cell development using easily accessible fibroblasts made to express Dll4. These cells, which are amenable for in vitro applications, can be further utilized in the design of individualized systems for T-cell production, with implications for the treatment of immunodeficiencies.

T cells typically differentiate via a series of coordinated steps within the highly specialized microenvironment of the thymus. Traditionally, human T-lymphopoiesis in vitro has been studied using the hybrid human/mouse fetal thymic organ culture system. Pioneering work by McCune et al. devised a method to examine human T cell development in vivo in relation to HIV-1 using the SCID/hu (thy/liv) model. This was followed by models that better reflected the ability of human hematopoietic cells to home and differentiate within the mouse host without human fetal tissues; however, human T cell development in these animals was poor. In this chapter, we outline a procedure to generate human progenitor T (proT) cells in vitro from umbilical cord blood-derived hematopoietic stem cells using the OP9-DL1 cell system; in addition, we describe the method used to examine the engraftment of in vitro-derived proT cells into immunodeficient mouse strains.

T cells are unique in that they begin their development as a progenitor within the bone marrow but complete their differentiation within the thymus. Furthermore, long-term T-lymphopoiesis requires a continuous supply of thymus-bound progenitors derived from the bone marrow. The critical role for T cells is clearly observed in individuals with genetic or acquired immunodeficiencies or those having undergone hematopoietic stem cell transplantation. Here, we review the work done by several groups aimed at characterizing the earliest T-lineage progenitors (ETPs), in mouse and human, found within the thymus, in addition to the long-sought after thymus-colonizing progenitor, which makes its journey from the bone marrow via the bloodstream into thymus. The characterization of these progenitors may herald therapeutic insight into the restoration of T cells in immunodeficient individuals.

Background T cell development occurs within the highly specialized thymus. Cytotoxic CD8 T cells are critical in adaptive immunity by targeting virally infected or tumor cells. In this study, we addressed whether functional CD8 T cells can be generated fully in vitro using human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) in coculture with OP9-DL1 cells. Results HSC/OP9-DL1 cocultures supported the differentiation of CD8 T cells, which were TCR/CD3hi CD27hi CD1aneg and thus phenotypically resembled mature functional CD8 single positive thymocytes. These in vitro-generated T cells also appeared to be conventional CD8 cells, as they expressed high levels of Eomes and low levels of Plzf, albeit not identical to ex vivo UCB CD8 T cells. Consistent with the phenotypic and molecular characterization, upon TCR-stimulation, in vitro-generated CD8 T cells proliferated, expressed activation markers (MHC-II, CD25, CD38), secreted IFN-γ and expressed Granzyme B, a cytotoxic T-cell effector molecule. Conclusion Taken together, the ability to direct human hematopoietic stem cell or T-progenitor cells towards a mature functional phenotype raises the possibility of establishing cell-based treatments for T-immunodeficiencies by rapidly restoring CD8 effector function, thereby mitigating the risks associated with opportunistic infections.

Human peripheral blood NK cells may be divided into two main subsets: CD56(bright)CD16(-) and CD56(dim)CD16(+). Since TGF-β is known to influence the development of many leukocyte lineages, its effects on NK cell differentiation either from human CD34(+)Lin(-) hematopoietic progenitor/stem cells in vitro or from peripheral blood NK cells were investigated. TGF-β represses development of NK cells from CD34(+) progenitors and inhibits differentiation of CD16(+) NK cells. Moreover, TGF-β also results in conversion of a minor fraction of CD56(bright)CD16(+) cells found in peripheral blood into CD56(bright)CD16(-) cells, highlighting a possible role of the former as a developmental intermediate and of TGF-β in influencing the genesis of NK subsets found in blood.

Traditionally, the study of human T cell development has relied on the availability of human and mouse thymic tissue. In this chapter, we outline a simple in vitro protocol for generating large numbers of human T-lineage cells from umbilical cord blood (CB)- derived hematopoietic stem cells (HSCs) using a bone marrow stromal cell line. This protocol is broken into three major steps: (1) the maintenance of a working stock of OP9 bone marrow stromal cells expressing the Notch receptor ligand Delta-like 1 (OP9- DL1), (2) the purification of human HSCs from umbilical CB, and (3) the initiation and maintenance/expansion of OP9-DL1 cocultures over time (see Fig. 1). The use of this system opens avenues for basic research as it equips us with a simple in vitro method for studying human T cell development.

Immunodeficient individuals are susceptible to opportunistic infection. While stem cell transplantation can restore a functional immune system, T cells are slow to recover and limited in eliciting adaptive immune responses. Approaches to selectively enhance T cell function have focused on boosting thymopoiesis to generate new T cells or expanding existing T cells. By taking advantage of the role of Notch signaling in T cell development, we have developed an in vitro system able to generate large numbers of progenitor T cells from human hematopoietic stem cells. Here, we discuss this in vitro system and its implications for the potential treatment of T cell immunodeficiency.