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.

Brief Research Report: Veterinary Student Perspective on COVID-19 and Veterinary Medicine.

COVID-19 has had significant effects on the field of veterinary medicine. Adaptation to pandemic-related and post-pandemic challenges requires engagement from all levels of the professional pipeline, including veterinary college students. Insights gained from this group may inform curriculum design, help the veterinary profession innovate, maximize opportunities for positive change, and avoid negative outcomes. The current study aimed to understand the potential impacts of the COVID-19 pandemic on veterinary medicine, as foreseen by second-year veterinary students in an online discussion during a public health course in the spring of 2020. Twenty-one percent of the 113 students agreed to participate in this qualitative research study. We used an inductive coding process and distilled the student responses into descriptive themes to capture diverse perspectives and understand possible post-pandemic pathways for the veterinary profession. Four themes emerged from the student discussion posts, describing how veterinarians might be affected by the COVID-19 pandemic: (1) economic and social impacts, (2) adapting to challenges, (3) collaborations to improve public health, and (4) disparities and diversity. These themes are a starting point for discussion and innovation as veterinarians plan for the post-pandemic world; further investigation will provide additional guidance for veterinary leaders.

Genetic Analysis of iNKT Cell Development and Function.

Natural killer T (NKT) cells are among the immediate and early responding immune cells and are important players in autoimmune diseases and tumor immunity. This unique subset of T cells shares properties of natural killer cells and T cells. Proper identification and characterization of NKT cell subsets is essential to understand the function and involvement of these understudied immune cells in various diseases. This review aims to summarize the known methods for identifying and characterizing NKT cells. NKT cells are divided into Type I (or invariant) and Type II, with either limited or broad TCR repertoires, respectively, that generally respond to glycolipids presented on the nonclassical MHC, CD1d. Type I NKT cells or invariant NKT cells (iNKT) are the most well studied and can be further subdivided into NKT1, NKT2, or NKT17 populations, classified based on their functional capacity. Conversely, less is known about Type II NKT cells because they have a more diverse TCR repertoire which make them hard to identify. However, genetic analyses have shed light on the development and function of all NKT subsets, which aids in their characterization. Further exploration of the role of NKT cells in various diseases will reveal the intricacies and importance of their novel functions.

Effect of Perfluorooctanesulfonic acid (PFOS) on immune cell development and function in mice.

Perfluoroctanesulfonate (PFOS) belongs to a larger family of compounds known as Per- and polyfluoroalkyl substances (PFAS). The strength of the carbon-fluorine bond makes PFOS extremely resistant to environmental degradation. Due to its persistent nature, research has been directed to elucidating possible health effects of PFOS on humans and laboratory animals. Here we have explored the effects of PFOS exposure on immune development and function in mice. We exposed adult mice to 3 and 1.5 µg/kg/day of PFOS for 2 and 4 weeks, respectively, and examined the effects of PFOS exposure on populations of T cells, B cells, and granulocytes. These doses of PFOS resulted in serum levels of approximately 100 ng/mL with no weight loss during exposure. We find that PFOS does not affect T-cell development during this time. However, while PFOS exposure reduced immune cell populations in some organs, it also led to an increase in the numbers of cells in others, suggesting possible relocalization of cells. We also examined the effect of PFOS on the response to influenza virus infection. We find that exposure to PFOS at 1.5 µg/kg/day of PFOS for 4 weeks does not affect weight loss or survival, nor is viral clearance affected. Analysis of antibody and T cell specific antiviral responses indicate that at this concentration, PFOS does not suppress the immune cell development or antigen specific immune response.

TCR Signal Strength and Antigen Affinity Regulate CD8+ Memory T Cells.

CD8+ T cells play a critical role in adaptive immunity, differentiating into CD8+ memory T cells that form the basis of protective cellular immunity. Vaccine efficacy is attributed to long-term protective immunity, and understanding the parameters that regulate development of CD8+ T cells is critical to the design of T cell-mediated vaccines. We show in this study using mouse models that two distinct parameters, TCR signal strength (regulated by the tyrosine kinase ITK) and Ag affinity, play important but separate roles in modulating the development of memory CD8+ T cells. Unexpectedly, our data reveal that reducing TCR signal strength along with reducing Ag affinity for the TCR leads to enhanced and accelerated development of CD8+ memory T cells. Additionally, TCR signal strength is able to regulate CD8+ T cell effector cytokine R production independent of TCR Ag affinity. Analysis of RNA-sequencing data reveals that genes for inflammatory cytokines/cytokine receptors are significantly altered upon changes in Ag affinity and TCR signal strength. Furthermore, our findings show that the inflammatory milieu is critical in regulating this TCR signal strength-mediated increase in memory development, as both CpG oligonucleotide treatment or cotransfer of wild-type and Itk-/- T cells eliminates the observed increase in memory cell formation. These findings suggest that TCR signal strength and Ag affinity independently contribute to CD8+ memory T cell development, which is modulated by inflammation, and suggest that manipulating TCR signal strength along with Ag affinity, may be used to tune the development of CD8+ memory T cells during vaccine development.

Interleukin-2-Inducible T-Cell Kinase Deficiency Impairs Early Pulmonary Protection Against Mycobacterium tuberculosis Infection.

Interleukin-2 (IL-2) inducible T-cell kinase (ITK) is a non-receptor tyrosine kinase highly expressed in T-cell lineages and regulates multiple aspects of T-cell development and function, mainly through its function downstream of the T-cell receptor. Itk deficiency can lead to CD4 lymphopenia and Epstein-Bar virus (EBV)-associated lymphoproliferation and recurrent pulmonary infections in humans. However, the role of the ITK signaling pathway in pulmonary responses in active tuberculosis due to Mtb infection is not known. We show here that human lungs with active tuberculosis exhibit altered T-cell receptor/ITK signaling and that Itk deficiency impaired early protection against Mtb in mice, accompanied by defective development of IL-17A-producing γδ T cells in the lungs. These findings have important implications of human genetics associated with susceptibility to Mtb due to altered immune responses and molecular signals modulating host immunity that controls Mtb activity. Enhancing ITK signaling pathways may be an alternative strategy to target Mtb infection, especially in cases with highly virulent strains in which IL-17A plays an essential protective role.

Past, Present, and Future of Rituximab-The World's First Oncology Monoclonal Antibody Therapy.

Rituximab is a chimeric mouse/human monoclonal antibody (mAb) therapy with binding specificity to CD20. It was the first therapeutic antibody approved for oncology patients and was the top-selling oncology drug for nearly a decade with sales reaching $8.58 billion in 2016. Since its initial approval in 1997, it has improved outcomes in all B-cell malignancies, including diffuse large B-cell lymphoma, follicular lymphoma, and chronic lymphocytic leukemia. Despite widespread use, most mechanistic data have been gathered from in vitro studies while the roles of the various response mechanisms in humans are still largely undetermined. Polymorphisms in Fc gamma receptor and complement protein genes have been implicated as potential predictors of differential response to rituximab, but have not yet shown sufficient influence to impact clinical decisions. Unlike most targeted therapies developed today, no known biomarkers to indicate target engagement/tumor response have been identified, aside from reduced tumor burden. The lack of companion biomarkers beyond CD20 itself has made it difficult to predict which patients will respond to any given anti-CD20 antibody. In the past decade, two new anti-CD20 antibodies have been approved: ofatumumab, which binds a distinct epitope of CD20, and obinutuzumab, a mAb derived from rituximab with modifications to the Fc portion and to its glycosylation. Both are fully humanized and have biological activity that is distinct from that of rituximab. In addition to these new anti-CD20 antibodies, another imminent change in targeted lymphoma treatment is the multitude of biosimilars that are becoming available as rituximab's patent expires. While the widespread use of rituximab itself will likely continue, its biosimilars will increase global access to the therapy. This review discusses current research into mechanisms and potential biomarkers of rituximab response, as well as its biosimilars and the newer CD20 binding mAb therapies. Increased ability to assess the effectiveness of rituximab in an individual patient, along with the availability of alternative anti-CD20 antibodies will likely lead to dramatic changes in how we use CD20 antibodies going forward.