Engineering human pluripotent stem cell lines to evade xenogeneic transplantation barriers.

Successful allogeneic human pluripotent stem cell (hPSC)-derived therapies must overcome immunological rejection by the recipient. To build reagents to define these barriers, we genetically ablated ß2M, TAP1, CIITA, CD74, MICA, and MICB to limit expression of HLA-I, HLA-II, and natural killer (NK) cell activating ligands in hPSCs. Transplantation of these cells that also expressed covalent single chain trimers of Qa1 and H2-Kb to inhibit NK cells and CD55, Crry, and CD59 to inhibit complement deposition led to persistent teratomas in wild-type mice. Transplantation of HLA-deficient hPSCs into mice genetically deficient in complement and depleted of NK cells also led to persistent teratomas. Thus, T cell, NK cell, and complement evasion are necessary to prevent immunological rejection of hPSCs and their progeny. These cells and versions expressing human orthologs of immune evasion factors can be used to define cell type-specific immune barriers and conduct preclinical testing in immunocompetent mouse models.

Engineering Human Pluripotent Stem Cell Lines to Evade Xenogeneic Transplantation Barriers.

Allogeneic human pluripotent stem cell (hPSC)-derived cells and tissues for therapeutic transplantation must necessarily overcome immunological rejection by the recipient. To define these barriers and to create cells capable of evading rejection for preclinical testing in immunocompetent mouse models, we genetically ablated ß2m, Tap1, Ciita, Cd74, Mica, and Micb to limit expression of HLA-I, HLA-II, and natural killer cell activating ligands in hPSCs. Though these and even unedited hPSCs readily formed teratomas in cord blood-humanized immunodeficient mice, grafts were rapidly rejected by immunocompetent wild-type mice. Transplantation of these cells that also expressed covalent single chain trimers of Qa1 and H2-Kb to inhibit natural killer cells and CD55, Crry, and CD59 to inhibit complement deposition led to persistent teratomas in wild-type mice. Expression of additional inhibitory factors such as CD24, CD47, and/or PD-L1 had no discernible impact on teratoma growth or persistence. Transplantation of HLA-deficient hPSCs into mice genetically deficient in complement and depleted of natural killer cells also led to persistent teratomas. Thus, T cell, NK cell, and complement evasion are necessary to prevent immunological rejection of hPSCs and their progeny. These cells and versions expressing human orthologs of immune evasion factors can be used to refine tissue- and cell type-specific immune barriers, and to conduct preclinical testing in immunocompetent mouse models.

Mitochondrial pyruvate metabolism and glutaminolysis toggle steady-state and emergency myelopoiesis.

To define the metabolic requirements of hematopoiesis, we examined blood lineages in mice conditionally deficient in genes required for long-chain fatty acid oxidation (Cpt2), glutaminolysis (Gls), or mitochondrial pyruvate import (Mpc2). Genetic ablation of Cpt2 or Gls minimally impacted most blood lineages. In contrast, deletion of Mpc2 led to a sharp decline in mature myeloid cells and a slower reduction in T cells, whereas other hematopoietic lineages were unaffected. Yet MPC2-deficient monocytes and neutrophils rapidly recovered due to a transient and specific increase in myeloid progenitor proliferation. Competitive bone marrow chimera and stable isotope tracing experiments demonstrated that this proliferative burst was progenitor intrinsic and accompanied by a metabolic switch to glutaminolysis. Myeloid recovery after loss of MPC2 or cyclophosphamide treatment was delayed in the absence of GLS. Reciprocally, MPC2 was not required for myeloid recovery after cyclophosphamide treatment. Thus, mitochondrial pyruvate metabolism maintains myelopoiesis under steady-state conditions, while glutaminolysis in progenitors promotes emergency myelopoiesis.

Orthogonal SARS-CoV-2 Serological Assays Enable Surveillance of Low-Prevalence Communities and Reveal Durable Humoral Immunity.

We conducted a serological study to define correlates of immunity against SARS-CoV-2. Compared to those with mild coronavirus disease 2019 (COVID-19) cases, individuals with severe disease exhibited elevated virus-neutralizing titers and antibodies against the nucleocapsid (N) and the receptor binding domain (RBD) of the spike protein. Age and sex played lesser roles. All cases, including asymptomatic individuals, seroconverted by 2 weeks after PCR confirmation. Spike RBD and S2 and neutralizing antibodies remained detectable through 5-7 months after onset, whereas α-N titers diminished. Testing 5,882 members of the local community revealed only 1 sample with seroreactivity to both RBD and S2 that lacked neutralizing antibodies. This fidelity could not be achieved with either RBD or S2 alone. Thus, inclusion of multiple independent assays improved the accuracy of antibody tests in low-seroprevalence communities and revealed differences in antibody kinetics depending on the antigen. We conclude that neutralizing antibodies are stably produced for at least 5-7 months after SARS-CoV-2 infection.

Detection, prevalence, and duration of humoral responses to SARS-CoV-2 under conditions of limited population exposure.

We conducted an extensive serological study to quantify population-level exposure and define correlates of immunity against SARS-CoV-2. We found that relative to mild COVID-19 cases, individuals with severe disease exhibited elevated authentic virus-neutralizing titers and antibody levels against nucleocapsid (N) and the receptor binding domain (RBD) and the S2 region of spike protein. Unlike disease severity, age and sex played lesser roles in serological responses. All cases, including asymptomatic individuals, seroconverted by 2 weeks post-PCR confirmation. RBD- and S2-specific and neutralizing antibody titers remained elevated and stable for at least 2-3 months post-onset, whereas those against N were more variable with rapid declines in many samples. Testing of 5882 self-recruited members of the local community demonstrated that 1.24% of individuals showed antibody reactivity to RBD. However, 18% (13/73) of these putative seropositive samples failed to neutralize authentic SARS-CoV-2 virus. Each of the neutralizing, but only 1 of the non-neutralizing samples, also displayed potent reactivity to S2. Thus, inclusion of multiple independent assays markedly improved the accuracy of antibody tests in low seroprevalence communities and revealed differences in antibody kinetics depending on the viral antigen. In contrast to other reports, we conclude that immunity is durable for at least several months after SARS-CoV-2 infection.

Sending Cancer into the Fetal Position.

Malignant cells gain the ability to self-renew and reacquire expression of proteins associated with embryonic development. In this issue of Cell Stem Cell, Kooreman et al. (2018) demonstrate that vaccination of mice with syngeneic inactivated iPSCs generates T cell immunity against embryonic antigens and provides resistance to several different types of cancers.

Mitochondrial Pyruvate Import Promotes Long-Term Survival of Antibody-Secreting Plasma Cells.

Durable antibody production after vaccination or infection is mediated by long-lived plasma cells (LLPCs). Pathways that specifically allow LLPCs to persist remain unknown. Through bioenergetic profiling, we found that human and mouse LLPCs could robustly engage pyruvate-dependent respiration, whereas their short-lived counterparts could not. LLPCs took up more glucose than did short-lived plasma cells (SLPCs) in vivo, and this glucose was essential for the generation of pyruvate. Glucose was primarily used to glycosylate antibodies, but glycolysis could be promoted by stimuli such as low ATP levels and the resultant pyruvate used for respiration by LLPCs. Deletion of Mpc2, which encodes an essential component of the mitochondrial pyruvate carrier, led to a progressive loss of LLPCs and of vaccine-specific antibodies in vivo. Thus, glucose uptake and mitochondrial pyruvate import prevent bioenergetic crises and allow LLPCs to persist. Immunizations that maximize these plasma cell metabolic properties might thus provide enduring antibody-mediated immunity.