Expansion Of Cell Populations Research Articles

Microcavity-assisted cloning (MAC) of hard-to-clone HepG2 cell lines: cloning made easy

Cloning is a key molecular biology procedure for obtaining a genetically homogenous population of organisms or cell lines. It requires the expansion of new cell populations starting from single genetically modified cells. Despite the technical progress, cloning of many cell lines remains difficult. Cloning often fails either due to the strenuous conditions associated with manipulating cells or because many cells don’t tolerate a single-cell state. Here we describe a new cloning method utilizing low adhesion microcavity plates. This new technique, named microcavity-assisted cloning (MAC) was developed to clone difficult-to-clone HepG2 cells. The clones were produced following CRISPR/Cas9 knockout of the GSTA1 gene by a random distribution of 200, 400, and 800 cells into 550 microcavities of a 24-well low adhesion plate originally designed for the culture of spheroids. The knockout of GSTA1 was verified at the protein level using Western blotting. The advantages of the MAC method are its low cost, ease of the procedure, and the possibility of scaling up the throughput and automatization.

Bone Marrow and Peripheral Blood Mononuclear Cell Phenotype Changes after Cultivation and Autologous Infusion in Patients with Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC) is a condition affecting the liver and immune system. In this study, the impact of autologous bone marrow-derived mononuclear cell (BM-MNC) transplantation on PBC patients was investigated. Sixteen eligible PBC patients participated at the National Scientific Medical Center in Astana, Kazakhstan, between 2017 and 2022, and BM-MNCs were harvested from their anterior iliac crest. After isolating and cultivating the BM-MNCs, they were infused back into the patient's peripheral veins. Changes in BM-MNC and peripheral blood mononuclear cell (PB-MNC) phenotypes were assessed before and after a 24-hour cultivation period and 72 hours post-transplantation. We monitored liver function parameters over 6-month intervals and conducted flow cytometry analysis to assess CD markers on BM-MNCs before and after cultivation and PB-MNCs before and after transplantation. Statistical analysis included the Friedman test for liver parameters and the Wilcoxon signed-rank test for BM-MNC and PB-MNC comparisons. Our findings revealed significant reductions in liver function tests after multiple transplantations. Flow cytometry analysis before and after a 24-hour culture and autologous BM-MNC infusion revealed the expansion of specific cell populations, with significant increases in CD3+, CD4+, CD16+, CD20+, CD25+, CD34+, CD105+, CD73+, СD117+, and CD34+populations, while CD4+25+, CD34+105+, and CD4+FOXP3+ populations decreased. Interestingly, a contradictory finding was observed with a decrease in bone marrow CD34+105+ cell lines (P=0.03) alongside an increase in peripheral CD34+105+ population (P=0.03). In summary, our study shows that BM-MNC transplantation in PBC patients leads to changes in immune cell populations and liver function. These findings suggest potential therapeutic applications of BM-MNC transplantation in managing PBC and offer insights into the dynamics of immune cells associated with this treatment approach.

Atypical cell cycle regulation promotes mammary stem cell expansion during mammary development and tumourigenesis

BackgroundThe cell cycle of mammary stem cells must be tightly regulated to ensure normal homeostasis of the mammary gland to prevent abnormal proliferation and susceptibility to tumorigenesis. The atypical cell cycle regulator, Spy1 can override cell cycle checkpoints, including those activated by the tumour suppressor p53 which mediates mammary stem cell homeostasis. Spy1 has also been shown to promote expansion of select stem cell populations in other developmental systems. Spy1 protein is elevated during proliferative stages of mammary gland development, is found at higher levels in human breast cancers, and promotes susceptibility to mammary tumourigenesis when combined with loss of p53. We hypothesized that Spy1 cooperates with loss of p53 to increase susceptibility to tumour initiation due to changes in susceptible mammary stem cell populations during development and drives the formation of more aggressive stem like tumours.MethodsUsing a transgenic mouse model driving expression of Spy1 within the mammary gland, mammary development and stemness were assessed. These mice were intercrossed with p53 null mice to study the tumourigenic properties of Spy1 driven p53 null tumours, as well as global changes in signaling via RNA sequencing analysis.ResultsWe show that elevated levels of Spy1 leads to expansion of mammary stem cells, even in the presence of p53, and an increase in mammary tumour formation. Spy1-driven tumours have an increased cancer stem cell population, decreased checkpoint signaling, and demonstrate an increase in therapy resistance. Loss of Spy1 decreases tumor onset and reduces the cancer stem cell population.ConclusionsThis data demonstrates the potential of Spy1 to expand mammary stem cell populations and contribute to the initiation and progression of aggressive, breast cancers with increased cancer stem cell populations.

A pathologically expanded, clonal lineage of IL-21-producing CD4+ T cells drives inflammatory neuropathy.

Inflammatory neuropathies, which include chronic inflammatory demyelinating polyneuropathy (CIDP) and Guillain Barré syndrome (GBS), result from autoimmune destruction of the PNS and are characterized by progressive weakness and sensory loss. CD4+ T cells play a key role in the autoimmune destruction of the PNS. Yet, key properties of pathogenic CD4+ T cells remain incompletely understood. Here, we used paired single-cell RNA-Seq (scRNA-Seq) and single-cell T cell receptor-sequencing (scTCR-Seq) of peripheral nerves from an inflammatory neuropathy mouse model to identify IL-21-expressing CD4+ T cells that were clonally expanded and multifunctional. These IL-21-expressing CD4+ T cells consisted of 2 transcriptionally distinct expanded cell populations, which expressed genes associated with T follicular helper (Tfh) and T peripheral helper (Tph) cell subsets. Remarkably, TCR clonotypes were shared between these 2 IL-21-expressing cell populations, suggesting a common lineage differentiation pathway. Finally, we demonstrated that IL-21 receptor-KO (IL-21R-KO) mice were protected from neuropathy development and had decreased immune infiltration into peripheral nerves. IL-21 signaling upregulated CXCR6, a chemokine receptor that promotes CD4+ T cell localization in peripheral nerves. Together, these findings point to IL-21 signaling, Tfh/Tph differentiation, and CXCR6-mediated cellular localization as potential therapeutic targets in inflammatory neuropathies.

Flt3 agonist enhances immunogenicity of arenavirus vector-based simian immunodeficiency virus vaccine in macaques.

Arenaviral vaccine vectors encoding simian immunodeficiency virus (SIV) immunogens are capable of inducing efficacious humoral and cellular immune responses in nonhuman primates. Several studies have evaluated the use of immune modulators to further enhance vaccine-induced T-cell responses. The hematopoietic growth factor Flt3L drives the expansion of various bone marrow progenitor populations, and administration of Flt3L was shown to promote expansion of dendritic cell populations in spleen and blood, which are targets of arenaviral vectors. Therefore, we evaluated the potential of Flt3 signaling to enhance the immunogenicity of arenaviral vaccines encoding SIV immunogens (SIVSME543 Gag, Env, and Pol) in rhesus macaques, with a rhesus-specific engineered Flt3L-Fc fusion protein. In healthy animals, administration of Flt3L-Fc led to a 10- to 100-fold increase in type 1 dendritic cells 7 days after dosing, with no antidrug antibody (ADA) generation after repeated dosing. We observed that administration of Flt3L-Fc fusion protein 7 days before arenaviral vaccine increased the frequency and activation of innate immune cells and enhanced T-cell activation with no treatment-related adverse events. Flt3L-Fc administration induced early innate immune activation, leading to a significant enhancement in magnitude, breadth, and polyfunctionality of vaccine-induced T-cell responses. The Flt3L-Fc enhancement in vaccine immunogenicity was comparable to a combination with αCTLA-4 and supports the use of safe and effective variants of Flt3L to augment therapeutic vaccine-induced T-cell responses.IMPORTANCEInduction of a robust human immunodeficiency virus (HIV)-specific CD4+ and CD8+ T-cell response through therapeutic vaccination is considered essential for HIV cure. Arenaviral vaccine vectors encoding simian immunodeficiency virus (SIV) immunogens have demonstrated strong immunogenicity and efficacy in nonhuman primates. Here, we demonstrate that the immunogenicity of arenaviral vectors encoding SIV immunogens can be enhanced by administration of Flt3L-Fc fusion protein 7 days before vaccination. Flt3L-Fc-mediated increase in dendritic cells led to robust improvements in vaccine-induced T- and B-cell responses compared with vaccine alone, and Flt3L-Fc dosing was not associated with any treatment-related adverse events. Importantly, immune modulation by either Flt3L-Fc or αCTLA-4 led to comparable enhancement in vaccine response. These results indicate that the addition of Flt3L-Fc fusion protein before vaccine administration can significantly enhance vaccine immunogenicity. Thus, safe and effective Flt3L variants could be utilized as part of a combination therapy for HIV cure.

Preclinical evaluation and first-in-dog clinical trials of PBMC-expanded natural killer cells for adoptive immunotherapy in dogs with cancer

BackgroundNatural killer (NK) cells are cytotoxic cells capable of recognizing heterogeneous cancer targets without prior sensitization, making them promising prospects for use in cellular immunotherapy. Companion dogs develop spontaneous cancers...

Abstract 5536: The role of p38 MAPK in the tumor-induced immune suppressive microenvironment in metastatic breast cancer

Abstract The tumor microenvironment (TME) in Metastatic Breast Cancer (MBC) is a major factor contributing to therapy resistance and suppression of antitumor immune response. Tumors promote expansion and recruitment of immune suppressive cells such as myeloid-derived suppressor cells (MDSCs) contributing to tumor invasion and suppressing anti-tumor T cells. Our prior work suggested a critical role of p38 MAPK in tumor-induced expansion and mobilization of myeloid cell populations thereby facilitating metastasis. The current study examined the role of p38 MAPK in tumor-induced changes in immune landscape and explored the mechanisms by which p38 mediates tumor-immune interactions. First, the contribution of T cells to anti-metastatic activity of p38 inhibitor (p38i) was addressed by depletion of CD8 T cells. Depletion of CD8+ T cells negated the effects of p38i on tumor growth and metastasis in the syngeneic 4T1 model. Next, we examined whether p38i exhibits a direct effect on T cells in vitro or in vivo. The in vitro assays showed that p38 blockade increased levels of CD44+ CD62L+ CD8+ T cells indicating enhancement of T cell differentiation. To determine the effects of p38 blockade on the immune landscape in vivo, single-cell RNA-seq and flow cytometry studies were performed in the 4T1 model with p38i or p38-deficient cells in which p38α/Mapk14 was inactivated by CRISPR/Cas9 (p38KO). The scRNA-seq data showed that p38 blockade increased the expression of T cell differentiation markers in the TME. Furthermore, p38 blockade significant decreased IL-6 signaling in myeloid cells, an important mediator of MDSC function. Assessment of myeloid cell populations showed a decreased expression of the immune suppressive signature. This observation was further validated in MDSCs isolated from the spleens of 4T1 tumor-bearing mice treated with p38i and in p38KO cohorts. Our prior work showed that p38i does not affect the generation of MDSCs in vitro. Therefore, we examined whether blockade of p38 affects the chemotactic capacity of conditioned media from MBC cells using mouse monocytes. The chemotaxis assays showed that p38i and p38KO largely reduced the ability of conditioned media to stimulate transwell migration of mouse monocytes. Notably, p38i and p38KO largely reduced expression of chemotactic cytokines in MBC cell models. Furthermore, p38i and p38KO reduced production and chemotactic ability of exosomes isolated from tumor cells. Together, these observations suggest that tumor p38 MAPK signaling promotes immune-suppressive TME and metastasis by facilitating expansion and mobilization of myeloid cell populations through the mechanisms involving production of exosomes and pro-myeloid chemokines/cytokines. This work highlights that p38 blockade can be utilized in combination with immune therapy or chemotherapy to enhance clinical benefits of immune therapy and reduce promyeloid effects of chemotherapy. Citation Format: Priyanka Rajan, Robert Zollo, Mackenzie Lieberman, Yanqi Guo, Mohammed Alruwaili, Mohammed Alqarni, Brian Morreale, Scott Olejniczak, Joseph Barbi, Scott Abrams, Andrei Bakin. The role of p38 MAPK in the tumor-induced immune suppressive microenvironment in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5536.

Abstract 3584: Context matters: Genomic profiling of PR isoform-specific actions in breast cancer stem cells reveals novel therapeutic insights for ER+ breast cancer

Abstract Exposure to progesterone is a recognized risk factor for breast cancer, and PGR polymorphisms are associated with various malignancies. The two progesterone receptor (PR) isoforms, full length PR-B and truncated PR-A, are expressed from the PGR gene in breast tissue and play crucial roles in normal physiology. An imbalance in the expression ratio of these isoforms, favoring increased levels of PR-A, is common in breast cancer and is associated with resistance to tamoxifen in Luminal A-type tumors. Notably, PRs have recently been implicated in promoting endocrine resistance and driving the expansion of cancer stem-like cell (CSC) populations implicated in early dissemination and maintenance of metastatic populations. Because prior gene expression studies were primarily conducted in 2D culture conditions, the isoform-specific molecular and epigenetic mechanisms underlying PR actions in advanced ER+ breast cancer remain elusive. Our goal was to define progesterone-driven gene expression in CSC cells by employing transcriptomic profiling of T47D cells cultivated in spheroid-culture conditions (3D) expressing exclusively PR-A or PR-B. Utilizing CUT&RUN, we have mapped the genomic binding sites unique to each PR isoform and identified shared sites. This genomic data has been integrated with phenotypic studies in cells expressing a single PR isoform. Our findings indicate that PR-A acts as a regulator of the cell cycle and senescence, while PR-B plays a pivotal role in cell metabolism and intracellular signaling. We find little overlap between PR-regulated gene sets derived from the same hormone-treated cells harvested from 2D relative to 3D conditions. Our genomic profiling of PRs in this 3D model system has unveiled novel strategies to combat CSC outgrowth and late recurrence in luminal breast cancer patients. This work has shifted our prior understanding of the role of PRs in gene regulation within the CSC population, and offers potential insights for therapeutic interventions in ER+ breast cancer. Citation Format: Noelle E. Gillis, Thu H. Truong, Jessica Finlay-Schultz, Carol A. Sartorius, Carol A. Lange. Context matters: Genomic profiling of PR isoform-specific actions in breast cancer stem cells reveals novel therapeutic insights for ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3584.

Amphiregulin Switches Progenitor Cell Fate for Lineage Commitment During Gastric Mucosal Regeneration

Background & AimsIsthmic progenitors, tissue-specific stem cells in the stomach corpus, maintain mucosal homeostasis by balancing between proliferation and differentiation to gastric epithelial lineages. The progenitor cells rapidly adopt an active state in response to mucosal injury. However, it remains unclear how the isthmic progenitor cell niche is controlled during the regeneration of damaged epithelium. MethodsWe recapitulated tissue recovery process after acute mucosal injury in the mouse stomach. BrdU incorporation was utilized to trace newly generated cells during the injury and recovery phases. To define epithelial lineage commitment process during recovery, we performed single cell RNA-sequencing (scRNA-seq) on epithelial cells from the mouse stomachs. We validated the effects of amphiregulin (AREG) on mucosal recovery, utilizing recombinant AREG treatment or AREG deficient mice. ResultsWe determined that an EGFR ligand, AREG, can control progenitor cell lineage commitment. Based on the identification of lineage-committed subpopulations in the corpus epithelium through scRNA-seq and BrdU incorporation, we showed that isthmic progenitors mainly transition into short-lived surface cell lineages, but are less frequently committed to long-lived parietal cell lineages in homeostasis. However, mucosal regeneration after damage directs the lineage commitment of isthmic progenitors towards parietal cell lineages. During recovery, AREG treatment promoted repopulation with parietal cells, while suppressing surface cell commitment of progenitors. In contrast, TGF-alpha did not alter parietal cell regeneration, but did induce expansion of surface cell populations. AREG deficiency impairs parietal cell regeneration, but increases surface cell commitment. ConclusionsThese data demonstrate that different EGF receptor ligands can distinctly regulate isthmic progenitor-driven mucosal regeneration and lineage commitment.

The Link between Autosomal Dominant Polycystic Kidney Disease and Chromosomal Instability: Exploring the Relationship.

In autosomal dominant polycystic kidney disease (ADPKD) with germline mutations in a PKD1 or PKD2 gene, innumerable cysts develop from tubules, and renal function deteriorates. Second-hit somatic mutations and renal tubular epithelial (RTE) cell death are crucial features of cyst initiation and disease progression. Here, we use established RTE lines and primary ADPKD cells with disease-associated PKD1 mutations to investigate genomic instability and DNA damage responses. We found that ADPKD cells suffer severe chromosome breakage, aneuploidy, heightened susceptibility to DNA damage, and delayed checkpoint activation. Immunohistochemical analyses of human kidneys corroborated observations in cultured cells. DNA damage sensors (ATM/ATR) were activated but did not localize at nuclear sites of damaged DNA and did not properly activate downstream transducers (CHK1/CHK2). ADPKD cells also had the ability to transform, as they achieved high saturation density and formed colonies in soft agar. Our studies indicate that defective DNA damage repair pathways and the somatic mutagenesis they cause contribute fundamentally to the pathogenesis of ADPKD. Acquired mutations may alternatively confer proliferative advantages to the clonally expanded cell populations or lead to apoptosis. Further understanding of the molecular details of aberrant DNA damage responses in ADPKD is ongoing and holds promise for targeted therapies.

Myeloid-derived suppressor cells from tumour-bearing mice induce the population expansion of CD19hiFcγRIIbhi regulatory B cells via PD-L1.

Myeloid-derived suppressor cells (MDSCs) increase in number and gain immunosuppressive functions in tumours and many other pathological conditions. MDSCs are characterized by their strong T-cell immunosuppressive capacity. The effects that MDSCs may have on B cells, especially within the tumour microenvironment, are less well understood. Here, we report that either monocytic MDSCs or polymorphonuclear MDSCs can promote increases in interleukin (IL)-10-expressing CD19hiFcγRIIbhi regulatory B cells in vitro and in vivo. Splenic transitional-1, -2, and -3 cells and marginal zone B cells, but not follicular B cells, differentiate into IL-10-expressing CD19hiFcγRIIbhi regulatory B cells. The adoptive transfer of CD19hiFcγRIIbhi regulatory B cells via tail vein injection can promote subcutaneous 3LL tumour growth in mice. The expression of programmed death-ligand 1 on MDSCs was found to be strongly associated with CD19hiFcγRIIbhi regulatory B cell population expansion. Furthermore, the frequency of circulating CD19+FcγRIIhi regulatory B cells was significantly increased in advanced-stage lung cancer patients. Our results unveil a critical role of MDSCs in regulatory B-cell differentiation and population expansion in lung cancer patients.

Dependence of human cell survival and proliferation on the CASP3 prodomain

Mechanisms that regulate cell survival and proliferation are important for both the development and homeostasis of normal tissue, and as well as for the emergence and expansion of malignant cell populations. Caspase-3 (CASP3) has long been recognized for its proteolytic role in orchestrating cell death-initiated pathways and related processes; however, whether CASP3 has other functions in mammalian cells that do not depend on its known catalytic activity have remained unknown. To investigate this possibility, we examined the biological and molecular consequences of reducing CASP3 levels in normal and transformed human cells using lentiviral-mediated short hairpin-based knockdown experiments in combination with approaches designed to test the potential rescue capability of different components of the CASP3 protein. The results showed that a ≥50% reduction in CASP3 levels rapidly and consistently arrested cell cycle progression and survival in all cell types tested. Mass spectrometry-based proteomic analyses and more specific flow cytometric measurements strongly implicated CASP3 as playing an essential role in regulating intracellular protein aggregate clearance. Intriguingly, the rescue experiments utilizing different forms of the CASP3 protein showed its prosurvival function and effective removal of protein aggregates did not require its well-known catalytic capability, and pinpointed the N-terminal prodomain of CASP3 as the exclusive component needed in a diversity of human cell types. These findings identify a new mechanism that regulates human cell survival and proliferation and thus expands the complexity of how these processes can be controlled.The graphical abstract illustrates the critical role of CASP3 for sustained proliferation and survival of human cells through the clearance of protein aggregates.

Hedgehog activation promotes osteogenic fates of growth plate resting zone chondrocytes through transient clonal competency.

The resting zone of the postnatal growth plate is organized by slow-cycling chondrocytes expressing parathyroid hormone-related protein (PTHrP), which include a subgroup of skeletal stem cells that contribute to the formation of columnar chondrocytes. The PTHrP-Indian hedgehog feedback regulation is essential for sustaining growth plate activities; however, molecular mechanisms regulating cell fates of PTHrP+ resting chondrocytes and their eventual transformation into osteoblasts remain largely undefined. Here, in a mouse model, we specifically activated Hedgehog signaling in PTHrP+ resting chondrocytes and traced the fate of their descendants using a tamoxifen-inducible Pthrp-creER line with patched-1-floxed and tdTomato reporter alleles. Hedgehog-activated PTHrP+ chondrocytes formed large, concentric, clonally expanded cell populations within the resting zone ("patched roses") and generated significantly wider columns of chondrocytes, resulting in hyperplasia of the growth plate. Interestingly, Hedgehog-activated PTHrP+ cell descendants migrated away from the growth plate and transformed into trabecular osteoblasts in the diaphyseal marrow space in the long term. Therefore, Hedgehog activation drives resting zone chondrocytes into transit-amplifying states as proliferating chondrocytes and eventually converts these cells into osteoblasts, unraveling a potentially novel Hedgehog-mediated mechanism that facilitates osteogenic cell fates of PTHrP+ skeletal stem cells.

DSP-0509, a TLR7 agonist, exerted synergistic anti-tumor immunity combined with various immune therapies through modulating diverse immune cells in cancer microenvironment.

Toll-like receptor 7 (TLR7) acts as a crucial component of the innate immune system. Upon TLR7 binding to its ligand, myeloid cells, including dendritic cells (DCs) and macrophages, are activated and play vital roles in initiating adaptive immunity. Consequently, TLR7 agonists have been employed in cancer immunotherapy. We have synthesized DSP-0509, a systemic injectable TLR7 agonist, and in this investigation, we examined the effects of DSP-0509 on tumor-infiltrating lymphocytes (TILs) utilizing single-cell RNA sequencing (scRNA-seq) in a mouse model bearing tumors. Our results demonstrated that DSP-0509 induced an expansion of immune cell populations, such as Natural Killer (NK) cells, CD4+ T cells, and CD4+ regulatory T cells (Tregs). Subsequently, we combined an Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor with DSP-0509 to enhance the antitumor efficacy by reducing Tregs, as DSP-0509 led to an increase in Treg presence within tumors. Our findings demonstrated that this combination therapy effectively reduced Treg infiltration within the tumor, leading to enhanced antitumor activity. To further prevent CD8+ T cell exhaustion, we combined DSP-0509 with an anti-PD-1 antibody and assessed the alterations in TILs using scRNA-seq. Our results indicated that the combination treatment significantly increased the cluster of CD8+ T cells expressing Gzmb, Prf1, Ctla4, and Icos, when compared to the administration of DSP-0509 alone. Additionally, we observed a marked rise in the M1-like macrophage cluster in the combination treatment group compared to the group receiving only DSP-0509. To validate the potential of modulating myeloid cells within the tumor to enhance antitumor efficacy, we combined DSP-0509 with an inhibitor targeting the receptor tyrosine kinase AXL. In bone marrow derived macrophages (BMDMs), the AXL inhibitor further amplified DSP-0509-stimulated TNFα secretion while reducing IL-10 secretion. As a final step, we evaluated the antitumor activity by combining DSP-0509 and the AXL inhibitor in an in vivo tumor model, which demonstrated increased efficacy. In summary, our study elucidated the effects of DSP-0509 on immune activity within the tumor microenvironment. These findings provided valuable insights that pave the way for the development of novel combination immunotherapy strategies.

Hematopoietic Stem and Progenitor Cell Trained Immunity Is Heterogeneous and Cross-Protective in a Mouse Model of M. Avium Infection

Recent studies suggest that infection epigenetically reprograms hematopoietic stem and progenitor cells (HSPCs) to enhance innate immune responses upon secondary infectious challenge, a process called “trained immunity.” Indeed, transfer of hematopoietic progenitors from mice infected with Mycobacterium bovis has been shown to confer long-lasting immunity against tuberculosis in the transplant recipients via an interferon gamma (IFNg) dependent process (Kaufmann 2018). However, much remains to be discovered about the breadth of protection conferred by trained immunity and the specific progenitor cell types responsible. We aimed to build upon our prior work examining the effects of IFNg signaling on hematopoietic stem cells to begin to answer these questions. We established a model of trained immunity in mice in response to Mycobacterium avium infection. Transfer of lineage-negative cKit+ hematopoietic progenitors from M. avium infected animals to naïve hosts was sufficient to confer protection against rechallenge in the transplant recipients up to 1 year post transplant. Macrophages derived from trained HSPCs demonstrated enhanced bacterial killing and metabolism, indicative of reprogramming in HSPCs that is conferred to innate immune cells. scRNA-seq analysis revealed that HSPCs activate IFNg-response genes heterogeneously upon primary challenge, suggesting that HSPC responses are neither monolithic nor confined to a specific cell type. Single cell transcriptomic analysis also revealed expansion of rare cell populations in the bone marrow. However, transfer of purified long-term HSCs was not sufficient to transmit trained immunity, suggesting cross-talk is required. To test whether IFNg alone is sufficient to induce trained immunity, mice were treated with a single dose of recombinant IFNg. Strikingly, this exposure was sufficient to induce enhanced bacterial killing and metabolism in bone marrow derived macrophages cultured from treated mice. To test the specificity of trained immunity, mice transplanted with influenza -trained HSPCs were challenged with M. avium infection and vice versa. Mice transplanted with influenza-trained HSPCs displayed enhanced immunity against M. avium challenge and vice versa, demonstrating cross protection against antigenically distinct pathogens. Together, these results indicate that primary infections can cause heterogeneous and long-standing HSPC responses, leading to bone marrow derived macrophages with enhanced killing capacity and cross-protectivity against alternative pathogens. These data will inform future studies to understand long term impacts of inflammatory stress on hematopoietic progenitors and the immune cells they produce.

TAF1, the Largest Subunit of Tfiid, Is Dispensable for Adult Hematopoiesis

Temporal and spatial control of gene expression is central to normal hematopoietic stem cell (HSC) biology. Many sequence-specific transcription factors have been identified as key regulators of HSC fate decision, while the function of general transcription factors in HSC behavior is poorly understood. We previously reported that TAF1, the largest subunit of the TFIID complex, plays a critical role in AML1-ETO driven acute myeloid leukemogenesis. To evaluate the function of TAF1 in normal fetal and adult hematopoiesis, we generated TAF1 conditional knockout (CKO) mice and identified an essential role of TAF1 in fetal erythropoiesis. Surprisingly, deletion of TAF1 in adult mice was not lethal to hematopoiesis; rather, we observed a marked expansion of hematopoietic stem and progenitor cell (HSPC) populations, with increased self-renewal and impaired differentiation capacity in these TAF1- null cells. Consistently, we found that TAF1-null HSPCs failed to up-regulate key differentiation-associated genes when induced to differentiate in vitro. Using a variety of genome-wide assays and biochemical approaches, we found that TAF1 loss not only disrupted TFIID complex formation and chromatin recruitment, but also reduced promoter accessibility, impaired RNA Polymerase II promoter recruitment and activity, as well as its promoter-proximal pausing mechanism. Thus, the effect of TAF1 loss on normal adult hematopoiesis primarily relates to the inability to upregulate genes involved in differentiation, while HSC self-renewal and the expression of self-renewal genes proceeds relatively normally.

Venetoclax Pre-Treatment of CLL/SLL Patients Enhances Autologous CAR T-Cells Efficacy

Introduction Venetoclax is an oral targeted therapy drug that is FDA-approved for the treatment of chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL/SLL) in both the frontline and relapsed/refractory (R/R) settings. While Venetoclax's dominant mechanism of action is the inhibition of Bcl-2 and thus, priming malignant CLL/SLL cells for apoptosis, there are emerging data highlighting the effects of Venetoclax on T-cell fitness/function. T-cell populations in CLL/SLL are known to be functionally impaired, in part due to abnormal expansion of T-regulatory cell (Treg) populations that blunt normal anti-tumoral cellular immune responses by CD8 + T-cells, Th1 Helper T-cells, and NK-cells and allows CLL/SLL cells to proliferate. Treg cell frequency has also been shown to directly correlate with CLL/SLL progression, and treatment of CLL/SLL results in lower circulating Treg populations. The unfavorable and generally anti-inflammatory tumoral microenvironment fostered by CLL/SLL cells is a major obstacle to the development of effective chimeric antigen receptor (CAR) T-cell options for this disease, for which there are currently no FDA-approved products despite more than a decade of investigation. Objective: In our current work, we studied the impact of Venetoclax on T-cell composition and function in serially collected specimens from patients with CLL/SLL; and evaluated the function of CAR T-cells manufactured from Venetoclax treated patients. Methods Peripheral blood mononuclear cells (PMBCs) and plasma was collected from CLL/SLL patients (N=5) who were treated with Venetoclax using the standard ramp up dosing-protocol. All procedures were approved by our IRB under BDR 164122. Samples were collected at pre-treatment (baseline) and several post-Venetoclax dosing timepoints (Day +1 day, day +7, and day +30). B-cells were isolated by negative selection using magnetic beads. CLL cells were the B-cells were exposed Venetoclax alone or in combination with several chemotherapy agents. Cell viability was determined by Cell Titer glo after 48hrs of drug exposure. In addition, PBMCs samples were analyzed using multicolor parameter flow cytometry to determine T-cell subsets composition including memory phenotype/stemness and others at each time point. Thorough serum cytokine (IL-15, IL-6, Il-2 and IL-10) analysis was conducted at each time point. Using baseline and 1-week post Venetoclax PBMCs samples, T-cells were activated, and autologous second generation CD19-directed CAR T-cells were manufactured. The efficacy of CARs were then tested at a 1:1 effector to target ratio against Raji and Raji 4RH cells for 24 hours and analyzed for cytotoxicity via flow cytometry. Results As expected Venetoclax was active in all samples tested. In addition, synergistic activity was observed when combined with chemotherapy agents or targeted agents (i.e. BRD-4 inhibitors). Regarding T-cell function, flow cytometry analysis at day +30 of Venetoclax therapy demonstrated that T-helper and T-cytotoxic T-cells displayed less exhaustion markers when compared to pre-Venetoclax samples (Baseline). Of interest, autologous αCD19 CAR T-cells manufactured 1 week following Venetoclax therapy induced higher cytotoxicity against Raji (rituximab chemotherapy sensitive cell lines) or Raji 4RH (rituximab-chemotherapy resistant cell lines) cells when compared to CAR T-cells generated from Venetoclax pre-treatment T-cells. Conclusion Our data suggests that Venetoclax has anti-tumor activities beyond direct CLL cytotoxicity. Venetoclax therapy appears to repair T-cell subsets in CLL patients. Perhaps by decreasing exhaustion markers in CD4/CD8 T-cells, Venetoclax potentiate the cytotoxicity of anti-CD19 CAR T cells against B-cell lymphoma. Further work is needed to fully elucidate the mechanism(s) by which Venetoclax modifies the fitness and function of T-cell subsets. These efforts may guide the development of combination strategies using BH-3 mimetics and T-cell engagers based therapeutic options for patients with CLL/SLL.

THU570 Isoform Specific Gene Regulation By Progesterone Receptors Drives Expansion Of Breast Cancer Stem Cell Expansion In 3D Cell Culture Models

Abstract Disclosure: N.E. Gillis: None. T.H. Truong: None. C.A. Lange: None. Exposure to progesterone is a risk factor for breast cancer, and numerous PGR polymorphisms are linked to breast, endometrial, and ovarian cancer. Two progesterone receptor (PR) isoforms are expressed from PGR in breast tissues: full-length PR-B and truncated PR-A. In normal breast epithelia, PR isoforms are equally expressed and mostly function as A:B heterodimers. An imbalance of this ratio, and thus a predominance of homodimers, is frequent in ER+ breast cancer. Luminal A-type estrogen receptor positive (ER+) tumors often exhibit an elevated PR-A:PR-B ratio that predicts resistance to tamoxifen. It has recently been shown that PRs promote endocrine resistance and drive expansion of breast cancer stem-like cell (CSC) populations. However, the isoform-specific molecular mechanisms of PR action in ER+ breast cancers remain undefined. The goal of our current work is to map the progesterone-driven gene expression in CSC cells through transcriptomic profiling of T47D cells grown in spheroid-culture conditions that express exclusively PR-A or PR-B. This transcriptomic data has been integrated with phenotypic studies of T47D and MCF7 cells that express a single isoform of PR. Our data shows PR-A is a regulator of the cell cycle and of senescence, while PR-B mediates cell metabolism and intracellular signaling. We further integrated our cell line data with transcriptomes from patient tumors that have been classified based on their PR-A:PR-B ratio and their response to treatment with anti-progestins. Our results are a first step towards re-defining the role of PRs in gene regulation within the CSC population. Presentation Date: Thursday, June 15, 2023

Impact of clonal hematopoiesis in COVID-19 patients at high risk for adverse clinical outcomes

PurposeClonal hematopoiesis (CH) describes the aging-associated expansion of mutant hematopoietic cell populations. In various cohorts, CH has been associated with increased morbidity and mortality from non-hematologic diseases such as cardiovascular disease and infections, including COVID-19. Comorbidities placing individuals at risk of complications from these disorders, such as diabetes, also increase in prevalence with age and frequently co-exist with CH. How CH interacts with other aging-associated comorbidities to impact human health remains unknown.MethodsWe assessed the impact of CH on the pre-existing end-organ damage and ultimate clinical outcomes among 242 patients hospitalized with COVID-19 at Michigan Medicine from March to June of 2020. In contrast to most previous studies, these patients skewed older with the majority having multiple comorbidities, which placed them at higher risk for end-organ damage and poor clinical outcomes.ResultsOverall CH was not significantly associated with increased COVID-19 mortality after controlling for other risk factors, although we did note a borderline-significant association specifically for non-DNMT3A CH mutations. In contrast, we observed a significant association between CH and pre-existing chronic kidney disease (CKD), which was strongest for DNMT3A mutant CH.ConclusionsThese data suggest that the clinical impact of CH is influenced by the specific gene(s) mutated and is further modified by other comorbidities and clinical risk factors frequently present in the elderly.

Abstract 28 A Novel Chemically Defined Culturing System to Support Hematopoietic Stem Cell Expansion

Abstract Introduction Hematopoietic stem cells (HSCs) are known to be multipotent and can reconstitute the entire blood and immune system following HSC transplantation (HSCT). However, the current HSCTs have many unresolved issues including difficulties in finding donors for bone marrow transplantation, preparing a sufficient HSC dose in cord blood transplantation, and the risk of graft-versus-host disease (GvHD) for all allogeneic HSCTs. In recent years, there have been substantial efforts to research and understand the biology of HSCs to develop cell therapy products that meet these challenges and provide an alternative to traditional HSCTs. Several culturing methods have been developed to stimulate HSC expansion ex vivo, but the use of biological materials, such as albumin, has not been ideal for pharmaceutical manufacturing, and it has been difficult inhibiting differentiation of HSCs into lineage-committed cells while culturing in a cytokine-filled medium. We have developed a novel albumin-free culturing system that supports proliferation of long-term HSCs ex vivo by replacing cytokines and albumin with chemical agonists and a caprolactam-based polymer. Objectives The objective of this study is to characterize our expanded cell population as part of an analysis to determine whether the population reconstitutes bone marrow and is therefore suitable for development of a cell therapy product. Methods Here we report the in vitro and in vivo characterization of proliferated HSCs using our novel culturing system compared with a nicotinamide system which is known to support HSC proliferation ex vivo. A colony forming assay, cell surface marker analysis using a flow cytometer, and in vivo repopulating analysis using the NOD/Shi-scid IL-2Rγnull (NOG) mouse were conducted. Results Surface marker analysis of the proliferated population shows more than 70% remaining as undifferentiated CD34+. Of which, a higher number of CD201+CD90+CD45RA- exists compared with the nicotinamide-cultured population. Colony forming unit assays indicate that our proliferated cell population contains a higher number of multipotent progenitors. The repopulating cell assay also shows that the proliferated cells have bone marrow reconstitution capability in bone marrow-ablated NOG mice. Discussion We believe that our chemically defined culturing system can selectively proliferate HSCs while restraining their differentiation and will contribute to the development of novel HSC therapy products.