Effects of Perfluorohexane Sulfonate Exposure on Immune Cell Populations in Naive Mice.

Perfluorohexane sulfonate (PFHxS) is a member of the per- and polyfluoroalkyls (PFAS) superfamily of molecules, characterized by their fluorinated carbon chains and use in a wide range of industrial applications. PFHxS and perfluorooctane sulfonate are able to accumulate in the environment and in humans with the approximated serum elimination half-life in the range of several years. More recently, some PFAS compounds have also been suggested as potential immunosuppressants. In this study, we analyze immune cell numbers in mice following 28-d repeated oral exposure to potassium PFHxS at 12, 120, 1,200, and 12,000 ng/kg/d, with resulting serum levels ranging up to ∼1,600 ng/ml, approximating ranges found in the general population and at higher levels in PFAS workers. The immunosuppressant cyclophosphamide was analyzed as a positive control. B cells, T cells, and granulocytes from the bone marrow, liver, spleen, lymph nodes, and thymus were evaluated. We found that at these exposures, there was no effect of PFHxS on major T or B cell populations, macrophages, dendritic cells, basophils, mast cells, eosinophils, neutrophils, or circulating Ab isotypes. By contrast, mice exposed to cyclophosphamide exhibited depletion of several granulocyte and T and B cell populations in the thymus, bone marrow, and spleen, as well as reductions in IgG1, IgG2b, IgG2c, IgG3, IgE, and IgM. These data indicate that exposures of up to 12,000 ng/kg of PFHxS for 28 d do not affect immune cell numbers in naive mice, which provides valuable information for assessing the risks and health influences of exposures to this compound.

The kinase ITK controls a Ca2+-mediated switch that balances TH17 and Treg cell differentiation.

The balance of proinflammatory T helper type 17 (TH17) and anti-inflammatory T regulatory (Treg) cells is crucial for immune homeostasis in health and disease. The differentiation of naïve CD4+ T cells into TH17 and Treg cells depends on T cell receptor (TCR) signaling mediated, in part, by interleukin-2-inducible T cell kinase (ITK), which stimulates mitogen-activated protein kinases (MAPKs) and Ca2+ signaling. Here, we report that, in the absence of ITK activity, naïve murine CD4+ T cells cultured under TH17-inducing conditions expressed the Treg transcription factor Foxp3 and did not develop into TH17 cells. Furthermore, ITK inhibition in vivo during allergic inflammation increased the Treg:TH17 ratio in the lung. These switched Foxp3+ Treg-like cells had suppressive function, and their transcriptomic profile resembled that of differentiated, induced Treg (iTreg) cells, but their chromatin accessibility profiles were intermediate between TH17 and iTreg cells. Like iTreg cells, switched Foxp3+ Treg-like cells had reductions in the expression of genes involved in mitochondrial oxidative phosphorylation and glycolysis, in the activation of the mechanistic target of rapamycin (mTOR) signaling pathway, and in the abundance of the TH17 pioneer transcription factor BATF. This ITK-dependent switch between TH17 and Treg cells depended on Ca2+ signaling but not on MAPKs. These findings suggest potential strategies for fine-tuning TCR signal strength through ITK to control the balance of TH17 and Treg cells.

Intranasal Vaccination with Recombinant TLR2-Active Outer Membrane Vesicles Containing Sequential M2e Epitopes Protects against Lethal Influenza a Challenge.

Influenza is a highly contagious respiratory disease, resulting in an estimated 3 to 5 million cases of severe illness annually. While most influenza vaccines are administered parenterally via injection, one shortcoming is that they do not generate a strong immune response at the site of infection, which can become important in a pandemic. Intranasal vaccines can generate both local and systemic protective immune responses, can reduce costs, and enhance ease of administration. Previous studies showed that parenterally administered outer membrane vesicles (OMVs) that carry sequences of the M2e protein (OMV-M2e) protect against influenza A/PR8 challenge in mice and ferrets. In the current study, we measured the effectiveness of the intranasal route of the OMV-M2e vaccine against the influenza A/PR8 strain in mice. We observed high anti-M2e IgG and IgA titers post-challenge in mice vaccinated intranasally with OMV-M2e. In addition, we observed a Th1/Tc1 bias in the vaccinated mice, and an increased Th17/Tc17 response, both of which correlated with survival to A/PR8 challenge and significantly lower lung viral titers. We conclude that the intranasal-route administration of the OMV-M2e vaccine is a promising approach toward generating protection against influenza A as it leads to an increased proinflammatory immune response correlating with survival to viral challenge.

PTEN loss in glioma cell lines leads to increased extracellular vesicles biogenesis and PD-L1 cargo in a PI3K-dependent manner.

Phosphatase and Tensin Homologue (PTEN) is one of the most frequently lost tumor suppressors in cancer and the predominant negative regulator of the PI3K/AKT signaling axis. A growing body of evidence has highlighted the loss of PTEN with immuno-modulatory functions including the upregulation of the programmed death ligand-1 (PD-L1), an altered tumor derived secretome that drives an immunosuppressive tumor immune microenvironment (TIME), and resistance to certain immunotherapies. Given their roles in immunosuppression and tumor growth, we examined whether the loss of PTEN would impact the biogenesis, cargo, and function of extracellular vesicles (EVs) in the context of the anti-tumor associated cytokine interferon-γ (IFN-γ). Through genetic and pharmacological approaches, we show that PD-L1 expression is regulated by JAK/STAT signaling, not PI3K signaling. Instead, we observe that PTEN loss positively upregulates cell surface levels of PD-L1 and enhances the biogenesis of EVs enriched with PD-L1 in a PI3K-dependent manner. We demonstrate that because of these changes, EVs derived from glioma cells lacking PTEN have a greater ability to suppress T cell receptor (TCR) signaling. Taken together, these findings provide important new insights into how the loss of PTEN can contribute to an immunosuppressive TIME, facilitate immune evasion, and highlight a novel role for PI3K signaling in the regulation of EV biogenesis and the cargo they contain.

Th2 and Th17-associated immunopathology following SARS-CoV-2 breakthrough infection in Spike-vaccinated ACE2-humanized mice.

Vaccines have demonstrated remarkable effectiveness in protecting against COVID-19; however, concerns regarding vaccine-associated enhanced respiratory diseases (VAERD) following breakthrough infections have emerged. Spike protein subunit vaccines for SARS-CoV-2 induce VAERD in hamsters, where aluminum adjuvants promote a Th2-biased immune response, leading to increased type 2 pulmonary inflammation in animals with breakthrough infections. To gain a deeper understanding of the potential risks and the underlying mechanisms of VAERD, we immunized ACE2-humanized mice with SARS-CoV-2 Spike protein adjuvanted with aluminum and CpG-ODN. Subsequently, we exposed them to increasing doses of SARS-CoV-2 to establish a breakthrough infection. The vaccine elicited robust neutralizing antibody responses, reduced viral titers, and enhanced host survival. However, following a breakthrough infection, vaccinated animals exhibited severe pulmonary immunopathology, characterized by a significant perivascular infiltration of eosinophils and CD4+ T cells, along with increased expression of Th2/Th17 cytokines. Intracellular flow cytometric analysis revealed a systemic Th17 inflammatory response, particularly pronounced in the lungs. Our data demonstrate that aluminum/CpG adjuvants induce strong antibody and Th1-associated immunity against COVID-19 but also prime a robust Th2/Th17 inflammatory response, which may contribute to the rapid onset of T cell-mediated pulmonary immunopathology following a breakthrough infection. These findings underscore the necessity for further research to unravel the complexities of VAERD in COVID-19 and to enhance vaccine formulations for broad protection and maximum safety.

Adaptive immune cells are necessary for SARS-CoV-2-induced pathology.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the ongoing global pandemic associated with morbidity and mortality in humans. Although disease severity correlates with immune dysregulation, the cellular mechanisms of inflammation and pathogenesis of COVID-19 remain relatively poorly understood. Here, we used mouse-adapted SARS-CoV-2 strain MA10 to investigate the role of adaptive immune cells in disease. We found that while infected wild-type mice lost ~10% weight by 3 to 4 days postinfection, rag-/- mice lacking B and T lymphocytes did not lose weight. Infected lungs at peak weight loss revealed lower pathology scores, fewer neutrophils, and lower interleukin-6 and tumor necrosis factor-α in rag-/- mice. Mice lacking αß T cells also had less severe weight loss, but adoptive transfer of T and B cells into rag-/- mice did not significantly change the response. Collectively, these findings suggest that while adaptive immune cells are important for clearing SARS-CoV-2 infection, this comes at the expense of increased inflammation and pathology.

Targeting kinase ITK treats autoimmune arthritis via orchestrating T cell differentiation and function.

IL-2 inducible T cell kinase (ITK) is critical in T helper subset differentiation and its inhibition has been suggested for the treatment of T cell-mediated inflammatory diseases. T follicular helper (Tfh), Th17 and regulatory T cells (Treg) also play important roles in the development of rheumatoid arthritis (RA), while the role of ITK in the development of RA and the intricate balance between effector T and regulatory T cells remains unclear. Here, we found that CD4+ T cells from RA patients presented with an elevated ITK activation. ITK inhibitor alleviated existing collagen-induced arthritis (CIA) and reduced antigen specific antibody production. Blocking ITK kinase activity interferes Tfh cell generation. Moreover, ITK inhibitor effectively rebalances Th17 and Treg cells by regulating Foxo1 translocation. Furthermore, we identified dihydroartemisinin (DHA) as a potential ITK inhibitor, which could inhibit PLC-γ1 phosphorylation and the progression of CIA by rebalancing Th17 and Treg cells. Out data imply that ITK activation is upregulated in RA patients, and therefore blocking ITK signal may provide an effective strategy to treat RA patients and highlight the role of ITK on the Tfh induction and RA progression.

Th2 and Th17-Associated Immunopathology Following SARS-CoV-2 Breakthrough Infection in Spike-Vaccinated ACE2-humanized Mice.

Vaccines have demonstrated remarkable effectiveness in protecting against COVID-19; however, concerns regarding vaccine-associated enhanced respiratory diseases (VAERD) following breakthrough infections have emerged. Spike protein subunit vaccines for SARS-CoV-2 induce VAERD in hamsters, where aluminum adjuvants promote a Th2-biased immune response, leading to increased type 2 pulmonary inflammation in animals with breakthrough infections. To gain a deeper understanding of the potential risks and the underlying mechanisms of VAERD, we immunized ACE2-humanized mice with SARS-CoV-2 Spike protein adjuvanted with aluminum and CpG-ODN. Subsequently, we exposed them to increasing doses of SARS-CoV-2 to establish a breakthrough infection. The vaccine elicited robust neutralizing antibody responses, reduced viral titers, and enhanced host survival. However, following a breakthrough infection, vaccinated animals exhibited severe pulmonary immunopathology, characterized by a significant perivascular infiltration of eosinophils and CD4+ T cells, along with increased expression of Th2/Th17 cytokines. Intracellular flow cytometric analysis revealed a systemic Th17 inflammatory response, particularly pronounced in the lungs. Our data demonstrate that aluminum/CpG adjuvants induce strong antibody and Th1-associated immunity against COVID-19 but also prime a robust Th2/Th17 inflammatory response, which may contribute to the rapid onset of T cell-mediated pulmonary immunopathology following a breakthrough infection. These findings underscore the necessity for further research to unravel the complexities of VAERD in COVID-19 and to enhance vaccine formulations for broad protection and maximum safety.

Endothelial SARS-CoV-2 infection is not the underlying cause of COVID-19-associated vascular pathology in mice.

Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. In the present study, we utilized a set of new mouse genetic tools developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID-19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells do not contribute significantly to the diverse vascular pathology associated with COVID-19.

Age-dependent acquisition of pathogenicity by SARS-CoV-2 Omicron BA.5.

Pathology studies of SARS-CoV-2 Omicron variants of concern (VOC) are challenged by the lack of pathogenic animal models. While Omicron BA.1 and BA.2 replicate in K18-hACE2 transgenic mice, they cause minimal to negligible morbidity and mortality, and less is known about more recent Omicron VOC. Here, we show that in contrast to Omicron BA.1, BA.5-infected mice exhibited high levels of morbidity and mortality, correlating with higher early viral loads. Neither Omicron BA.1 nor BA.5 replicated in brains, unlike most prior VOC. Only Omicron BA.5-infected mice exhibited substantial weight loss, high pathology scores in lungs, and high levels of inflammatory cells and cytokines in bronchoalveolar lavage fluid, and 5- to 8-month-old mice exhibited 100% fatality. These results identify a rodent model for pathogenesis or antiviral countermeasure studies for circulating SARS-CoV-2 Omicron BA.5. Further, differences in morbidity and mortality between Omicron BA.1 and BA.5 provide a model for understanding viral determinants of pathogenicity.

Endothelial SARS-CoV-2 infection is not the underlying cause of COVID19-associated vascular pathology in mice.

Endothelial damage and vascular pathology have been recognized as major features of COVID-19 since the beginning of the pandemic. Two main theories regarding how Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) damages endothelial cells and causes vascular pathology have been proposed: direct viral infection of endothelial cells or indirect damage mediated by circulating inflammatory molecules and immune mechanisms. However, these proposed mechanisms remain largely untested in vivo. Here, we utilized a set of new mouse genetic tools 1 developed in our lab to test both the necessity and sufficiency of endothelial human angiotensin-converting enzyme 2 (hACE2) in COVID19 pathogenesis. Our results demonstrate that endothelial ACE2 and direct infection of vascular endothelial cells does not contribute significantly to the diverse vascular pathology associated with COVID-19.

Monoclonal antibodies binding to different epitopes of CD20 differentially sensitize DLBCL to different classes of chemotherapy.

Introduction: Rituximab (R), an anti-CD20 monoclonal antibody (mAb) and the world's first approved antibody for oncology patients, was combined with the CHOP chemotherapy regimen and markedly improved the prognosis of all B- cell-derived lymphomas, the most common hematological malignancy worldwide. However, there is a 35% disease recurrence with no advancement in the first-line treatment since R was combined with the archetypal CHOP chemotherapy regimen nearly 30 years ago. There is evidence that R synergizes with chemotherapy, but the pharmacological interactions between R and CHOP or between newer anti-CD20 mAbs and CHOP remain largely unexplored. Methods: We used in vitro models to score pharmacological interactions between R and CHOP across various lymphoma cell lines. We compared these pharmacological interactions to ofatumumab, a second-generation anti-CD20 mAb, and CHOP. Lastly, we used RNA-sequencing to characterize the transcriptional profiles induced by these two antibodies and potential molecular pathways that mediate their different effects. Results: We discovered vast heterogeneity in the pharmacological interactions between R and CHOP in a way not predicted by the current clinical classification. We then discovered that R and ofatumumab differentially synergize with the cytotoxic and cytostatic capabilities of CHOP in separate distinct subsets of B-cell lymphoma cell lines, thereby expanding favorable immunochemotherapy interactions across a greater range of cell lines beyond those induced by R-CHOP. Lastly, we discovered these two mAbs differentially modulate genes enriched in the JNK and p38 MAPK family, which regulates apoptosis and proliferation. Discussion: Our findings were completely unexpected because these mAbs were long considered to be biological and clinical equivalents but, in practice, may perform better than the other in a patient-specific manner. This finding may have immediate clinical significance because both immunochemotherapy combinations are already FDA-approved with no difference in toxicity across phase I, II, and III clinical trials. Therefore, this finding could inform a new precision medicine strategy to provide additional therapeutic benefit to patients with B-cell lymphoma using immunochemotherapy combinations that already meet the clinical standard of care.

Juneteenth in STEMM and the barriers to equitable science.

We are 52 Black scientists. Here, we establish the context of Juneteenth in STEMM and discuss the barriers Black scientists face, the struggles they endure, and the lack of recognition they receive. We review racism's history in science and provide institutional-level solutions to reduce the burdens on Black scientists.

ITK signaling regulates a switch between T helper 17 and T regulatory cell lineages via a calcium-mediated pathway.

The balance of pro-inflammatory T helper type 17 (Th17) and anti-inflammatory T regulatory (Treg) cells is crucial in maintaining immune homeostasis in health and disease conditions. Differentiation of naïve CD4+ T cells into Th17/Treg cells is dependent upon T cell receptor (TCR) activation and cytokine signaling, which includes the kinase ITK. Signals from ITK can regulate the differentiation of Th17 and Treg cell fate choice, however, the mechanism remains to be fully understood. We report here that in the absence of ITK activity, instead of developing into Th17 cells under Th17 conditions, naïve CD4+ T cells switch to cells expressing the Treg marker Foxp3 (Forkhead box P3). These switched Foxp3+ Treg like cells retain suppressive function and resemble differentiated induced Tregs in their transcriptomic profile, although their chromatin accessibility profiles are intermediate between Th17 and induced Tregs cells. Generation of the switched Foxp3+ Treg like cells was associated with reduced expression of molecules involved in mitochondrial oxidative phosphorylation and glycolysis, with reduced activation of the mTOR signaling pathway, and reduced expression of BATF. This ITK dependent switch between Th17 and Treg cells was reversed by increasing intracellular calcium. These findings suggest potential strategies for fine tune the TCR signal strength via ITK to regulate the balance of Th17/Treg cells.

Degrading the signal amplifier: ITK as a target for targeted protein degradation.

In this issue of Cell Chemical Biology, Jiang and colleagues show for the first time that the Tec kinase ITK can be targeted using PROTAC approaches. This new modality has implications for the treatment of T cell lymphomas, but also potentially for the treatment of T cell-mediated inflammatory diseases, that depend on ITK signaling.

Effective differentiation of double negative thymocytes requires high fidelity replication of mitochondrial DNA in an age dependent manner.

One of the most proliferative periods for T cells occurs during their development in the thymus. Increased DNA replication can result in increased DNA mutations in the nuclear genome, but also in mitochondrial genomes. A high frequency of mitochondrial DNA mutations can lead to abnormal mitochondrial function and have negative implications on human health. Furthermore, aging is accompanied by an increase in such mutations through oxidative damage and replication errors. Increased mitochondrial DNA mutations cause loss of mitochondrial protein function, and decrease energy production, substrates, and metabolites. Here we have evaluated the effect of increased mitochondrial DNA mutations on T cell development in the thymus. Using mice carrying a mutant mitochondrial DNA polymerase γ (PolG) that causes increased mitochondrial DNA mutations, we show that high fidelity replication of mitochondrial DNA is pivotal for proper T cell development. Reducing the fidelity of mitochondrial DNA replication results in a premature age-dependent reduction in the total number of CD4/CD8 double negative and double positive thymocytes. Analysis of mitochondrial density in thymocyte subpopulations suggests that this may be due to reduced proliferation in specific double negative stages. Taken together, this work suggests that T cell development is regulated by the ability of mitochondria to faithfully replicate their DNA.

Effects of PFOS and cyclophosphamide exposure on immune homeostasis in mice.

Perfluorooctane sulfonic acid (PFOS) is member of a class of molecules with fluorinated carbon chains known as polyfluoroalkyls. PFOS have been used to produce a variety of industry and comsumer uses. However, a significant concern is that it accumulates in the environment, including in animals and humans, and that it is a potential immunosuppressant. Here we analyze immune homeostasis in mice following chronic exposure to PFOS at levels up to those historically found in PFOS manufacturing workers. Mice were exposed to 0.15, 1.5, 15, or 50 µg /kg of PFOS for 28 days, after which, B cells, T cells, and granulocytes from the bone marrow, liver, spleen, lymph nodes, and thymus were evaluated. We find that at these exposures, there was no effect of PFOS on major T- or B-cell populations, macrophages, dendritic cells, basophils, mast cells, eosinophils, neutrophils, serum antibodies or select serum cytokines. By contrast, mice exposed the known immunosuppressant cyclophosphamide, which was given at 40 mg/kg for four days, exhibited depletion of several granulocyte, T- and B-cell populations of the thymus, bone marrow, and spleen, as well as circulating IgM and IgE antibodies. These data indicate that exposures of up to 50 µg /kg of PFOS for 28 days does not affect immune homeostasis in mice.

Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection.

Angiotensin-converting enzyme 2 (ACE2) is the cell-surface receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). While its central role in Coronavirus Disease 2019 (COVID-19) pathogenesis is indisputable, there remains significant debate regarding the role of this transmembrane carboxypeptidase in the disease course. These include the role of soluble versus membrane-bound ACE2, as well as ACE2-independent mechanisms that may contribute to viral spread. Testing these roles requires in vivo models. Here, we report humanized ACE2-floxed mice in which hACE2 is expressed from the mouse Ace2 locus in a manner that confers lethal disease and permits cell-specific, Cre-mediated loss of function, and LSL-hACE2 mice in which hACE2 is expressed from the Rosa26 locus enabling cell-specific, Cre-mediated gain of function. Following exposure to SARS-CoV-2, hACE2-floxed mice experienced lethal cachexia, pulmonary infiltrates, intravascular thrombosis and hypoxemia-hallmarks of severe COVID-19. Cre-mediated loss and gain of hACE2 demonstrate that neuronal infection confers lethal cachexia, hypoxemia, and respiratory failure in the absence of lung epithelial infection. In this series of genetic experiments, we demonstrate that ACE2 is absolutely and cell-autonomously required for SARS-CoV-2 infection in the olfactory epithelium, brain, and lung across diverse cell types. Therapies inhibiting or blocking ACE2 at these different sites are likely to be an effective strategy towards preventing severe COVID-19.