Naïve and memory T cells co-exist in the peripheral T cell pool, but the cellular mechanisms that maintain the balance and homeostasis of these two populations remain mostly unclear. To address this question, here, we assessed homeostatic proliferation and repopulation kinetics of adoptively transferred naïve and memory T cells in lymphopenic host mice. We identified distinct kinetics of proliferation and tissue-distribution between naïve and memory donor T cells, which resulted in the occupancy of the peripheral T cell pool by mostly naïve-origin T cells in short term (4 weeks). To explain this finding, we assessed utilization of the homeostatic cytokines IL-7 and IL-15 by naïve and memory T cells. We found different efficiencies of IL-7 signaling between naïve and memory T cells, where memory T cells expressed larger amounts of IL-7R but were significantly less efficient in activation of STAT5 that is downstream of IL-7 signaling. Nonetheless, memory T cells were superior in long-term repopulation of the peripheral T cell pool, presumably, because they preferentially migrated into non-lymphoid tissues upon adoptive transfer and then additionally utilized tissue IL-15 for rapid expansion. Consequently, co-utilization of IL-7 and IL-15 provides memory T cells a long-term survival advantage. We consider this mechanism important, as it permits the memory T cell population to be maintained in face of constant influx of naïve T cells to the peripheral T cell pool and under competing conditions for survival cytokines.
Deuterated water (2H2O) is a label commonly used for safe quantitative measurement of deuterium enrichment into DNA of proliferating cells. More recently, it has been used for labeling proteins and other biomolecules. Our in vitro - in vivo research reports important stable isotopic labeling enrichment differences into the DNA nucleosides and their isotopologues (e.g. deoxyadenosine (dA) M + 1, dA M + 2, dA M + 3), as well as tumor cell proliferation effects for various forms of commercially available stable heavy water (2H2O, H218O, and 2H218O). Using an in vitro mouse thymus tumor cell line, we determined that H218O provides superior DNA labeling enrichment quantitation, as measured by GC-positive chemical ionization (PCI)-MS/MS. In addition, at higher but physiologically relevant doses, both 2H218O and 2H2O down modulated mouse thymus tumor cell proliferation, whereas H218O water had no observable effects on cell proliferation. The in vivo labeling studies, where normal mouse bone marrow cells (i.e. high turnover) were evaluated post labeling, demonstrated DNA enrichments concordant with measurements from the in vitro studies. Our research also reports a headspace-GC-NCI-MS method, which rapidly and quantitatively measures stable heavy water levels in total body water.
Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplant (AHSCT). Although current therapies largely target T cell proliferation, in vivo T cell kinetics of division, death and trafficking have been difficult to evaluate. We employed deuterated water labeling with subsequent GC-MS/MS enrichment detection to measure in vivo T cell kinetics after AHSCT in a mouse model of cGVHD. We investigated CD4 T cell sub-population kinetics of cell division, death and trafficking, in non-target and target tissues of cGVHD. Early in the disease course, spleens of AHSCT recipients showed a high number of CD4+ T regulatory (Treg) and T effector memory (Tem) cells. In vivo cell division kinetics of these two cell types were high and similar to each other, and slowed as cGVHD progressed. Meanwhile, Tem cell loss from the spleen was significantly faster than that of Treg cells, likely as a result of increased trafficking. In support of trafficking was the finding of elevated Tem numbers in peripheral blood of AHSCT recipients despite a relative lymphopenia early in the disease with subsequent disappearance from the circulation once cGVHD was clinically evident. Furthermore, overall Tem cell numbers in the host were significantly higher than the number present in the graft, but distributed to target rather than non-target tissues. Target sites of cGVHD (skin, gut and liver) had several fold greater number of Tem cells compared to controls both early and late in disease. The number of Tem cells in target sites was maintained as cGVHD progressed, and the Treg to Tem ratio in target tissues was significantly lower than that of the controls at all times. Cell division of Tem and Treg cells was high in the liver, and remained high for the Treg cells as disease progressed. Meanwhile, Treg cells underwent faster loss from the liver, likely due to cell death given that the total number of Treg cells did not change. Our findings shed light on in vivo behavior of Treg and Tem cells, T cell subtypes critical to the biology of cGVHD. Furthermore, our methodology will be translated to investigate in vivo T cell kinetics in patients undergoing AHSCT as deuterated water labeling is safe, not toxic and non-radioactive.
Abstract Early events in the host response to SARS-CoV-2 are thought to play a major role in determining disease severity. During pulmonary infection, the virus encounters both myeloid and epithelioid lineage cells that can either support or restrict pathogen replication as well as respond with host protective versus detrimental mediators. In addition to providing partial protection against pediatric tuberculosis, vaccination with bacille Calmette-Guérin (BCG) has been reported to confer non-specific resistance to unrelated pulmonary pathogens, a phenomenon attributed to the induction of long-lasting alterations within the myeloid cell compartment. Here we demonstrate that prior intravenous, but not subcutaneous, administration of BCG protects human-ACE2 transgenic mice against lethal challenge with SARS-CoV-2 and results in reduced viral loads in non-transgenic animals infected with an alpha variant. The observed increase in host resistance was associated with reductions in SARS-CoV-2-induced tissue pathology, inflammatory cell recruitment and cytokine production that multivariate analysis revealed to be only partially related to diminished viral load. We propose that this protection stems from BCG-induced alterations in the composition and function of the pulmonary cellular compartment that impact the innate response to the virus and the ensuing immunopathology.
Severity of COVID-19 is affected by multiple factors; however, it is not understood how the inflammatory milieu of the lung at the time of SARS-CoV-2 exposure affects the control of viral replication. Here, we demonstrate that immune events in the mouse lung closely preceding SARS-CoV-2 infection affect viral control and identify innate immune pathways that limit viral replication. Pulmonary inflammatory stimuli including resolved, antecedent respiratory infections with Staphylococcus aureus or influenza, ongoing pulmonary Mycobacterium tuberculosis infection, ovalbumin/alum-induced asthma, or airway administration of TLR ligands and recombinant cytokines all establish an antiviral state in the lung that restricts SARS-CoV-2 replication. In addition to antiviral type I interferons, TNFα and IL-1 potently precondition the lung for enhanced viral control. Our work shows that SARS-CoV-2 may benefit from an immunologically quiescent lung microenvironment and suggests that heterogeneity in pulmonary inflammation preceding SARS-CoV-2 exposure may contribute to variability in disease outcomes.
It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.
Abstract Recent thymic emigrants (RTE) are newly generated T cells that are necessary to replenish T cell antigen receptor diversity and rejuvenate the peripheral naïve T cell pool. Both naïve T cells and RTE require IL-7 to survive in the periphery, but it is not known how RTE successfully compete with resident T cells for IL-7 signaling. To address this question, we assessed IL-7 receptor (IL-7Ra) expression on RTE, and found that RTE expresse substantially lower levels of IL-7Ra than naïve T cells. Because IL-7 signaling downregulates expression of its own receptor, lower IL-7Ra expression suggest that RTE are potentially more responsive to IL-7 than naïve T cells. Indeed, in vitro IL-7 stimulation showed significantly increased STAT5 phosphorylation in RTE compared to naïve T cells. To understand the molecular basis for increased IL-7 responsiveness, we assessed STAT5 phosphorylation and IL-7R expression upon IL-7 re-stimulation, and found that RTE are significantly more responsive to IL-7 re-stimulation. Contrary, adoptive transfer of RTE cells into lymphopenic host mice resulted in less efficient proliferation and expansion than of naïve donor T cells, which was not rescued by increasing in vivo IL-7 levels by implanting IL-7 osmotic pumps. Collectively, these data indicate that increased IL-7 responsiveness in RTE maximizes their survival while decreasing their proliferative capacity such as under lymphopenic conditions.
Abstract Although histone H3 lysine 27 trimethylation (H3K27Me3) is associated with gene silencing, whether H3K27Me3 demethylation affects transcription and cell differentiation in vivo has remained elusive. To investigate this, we conditionally inactivated the two H3K27Me3 demethylases, Jmjd3 and Utx, in non-dividing intrathymic CD4 + T-cell precursors. Here we show that both enzymes redundantly promote H3K27Me3 removal at, and expression of, a specific subset of genes involved in terminal thymocyte differentiation, especially S1pr1 , encoding a sphingosine-phosphate receptor required for thymocyte egress. Thymocyte expression of S1pr1 was not rescued in Jmjd3- and Utx-deficient male mice, which carry the catalytically inactive Utx homolog Uty, supporting the conclusion that it requires H3K27Me3 demethylase activity. These findings demonstrate that Jmjd3 and Utx are required for T-cell development, and point to a requirement for their H3K27Me3 demethylase activity in cell differentiation.
SARS-CoV-2 infection leads to vastly divergent clinical outcomes ranging from asymptomatic infection to fatal disease. Co-morbidities, sex, age, host genetics and vaccine status are known to affect disease severity. Yet, how the inflammatory milieu of the lung at the time of SARS-CoV-2 exposure impacts the control of viral replication remains poorly understood. We demonstrate here that immune events in the mouse lung closely preceding SARS-CoV-2 infection significantly impact viral control and we identify key innate immune pathways required to limit viral replication. A diverse set of pulmonary inflammatory stimuli, including resolved antecedent respiratory infections with
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