Follicular helper T (Tfh) cells have been implicated in controlling rejection after allogeneic kidney transplantation, but the precise subsets, origins, and functions of Tfh cells in this process have not been fully characterized. Here we show that a subset of effector Tfh cells marked by previous IL-21 production is potently induced during allogeneic kidney transplantation and is inhibited by immunosuppressive agents. Single-cell RNA-Seq revealed that these lymph node (LN) effector Tfh cells have transcriptional and clonal overlap with IL-21-producing kidney-infiltrating Tfh cells, implicating common origins and developmental trajectories. To investigate the precise functions of IL-21-producing effector Tfh cells in LNs and allografts, we used a mouse model to selectively eliminate these cells and assessed allogeneic B cell clonal dynamics using a single B cell culture system. We found that IL-21-producing effector Tfh cells were essential for transplant rejection by regulating donor-specific germinal center B cell clonal dynamics both systemically in the draining LN and locally within kidney grafts. Thus, IL-21-producing effector Tfh cells have multifaceted roles in Ab-mediated rejection after kidney transplantation by promoting B cell alloimmunity.

Follicular helper T (Tfh) cells are essential for the formation of high affinity antibodies after vaccination or infection. Although the signals responsible for initiating Tfh differentiation from naïve T cells have been studied, the signals controlling sequential developmental stages culminating in optimal effector function are not well understood. Here we use fate mapping strategies for the cytokine IL-21 to uncover sequential developmental stages of Tfh differentiation including a progenitor-like stage, a fully developed effector stage and a post-effector Tfh stage that maintains transcriptional and epigenetic features without IL-21 production. We find that progression through these stages are controlled intrinsically by the transcription factor FoxP1 and extrinsically by follicular regulatory T cells. Through selective deletion of Tfh stages, we show that these cells control antibody dynamics during distinct stages of the germinal center reaction in response to a SARS-CoV-2 vaccine. Together, these studies demonstrate the sequential phases of Tfh development and how they promote humoral immunity.

Background. Following allogeneic kidney transplantation, a substantial proportion of graft loss is attributed to the formation of donor-specific antibodies and antibody-mediated rejection. B cells infiltrate kidney grafts during antibody-mediated rejection; however, the origins, repertoires, and functions of these intrarenal B cells remain elusive. Methods. Here, we use murine allogeneic kidney transplant models to study the origins, transcriptional programming and B cell receptor repertoire of intragraft B cells, and in vitro stimulation assays to evaluate the ability of intragraft B cells to promote CD4+ T cell expansion. Results. B cells infiltrate kidney grafts in settings of allogeneic, but not syngeneic, transplantation. Intragraft B cells have characteristics of activation but are transcriptionally distinct from germinal center B cells and resemble innate-like B cells. B cell receptor sequencing demonstrates that the majority of intragraft B cells do not originate from lymph node germinal center B cells and are largely germline. Class-switched intragraft B cells are rare but can be donor-specific and produce IgG capable of binding to the kidney allograft. Lastly, intrarenal B cells are capable of stimulating naive T cells but have an altered ability to promote T follicular helper cell expansion. Conclusions. Together, these data demonstrate that intrarenal B cells during transplant rejection are transcriptionally distinct from lymph node B cells.

Bechu, Elsa The Role of Single Nucleotide Polymorphisms Causing a Dysregulation of the HPA Axis on the Incidence of Depression. Department of Biological Sciences, 2021. Advisor: Brian Cohen Depression is one of the most prevalent diseases worldwide, afflicting approximately 17 million adults in the US in 2019. A prominent causal factor of depression is dysregulation of our body’s response to stress when exposed to continuous stressors over long periods of time. Stress is regulated by the Hypothalamic-Pituitary-Adrenal (HPA) axis, with cortisol as its effector hormone. The effects of cortisol are exerted through the glucocorticoid and mineralocorticoid receptors (GR and MR, respectively) in the body and the brain, respectively. The HPA axis is regulated by a negative feedback loop where activation of the GR by circulating cortisol inhibits the production of additional cortisol. Our lab investigated the role of single nucleotide polymorphisms (SNPs), or mutations, in the genes of the MR, GR, and associated regulatory proteins that are hypothesized to be involved in the dysregulation of function leading to hypersensitivity or resistance to cortisol in the HPA axis. Previous literature has demonstrated that prominent GR and MR SNPs correlate with higher incidence of depression and/or depressive symptoms. Our study continued this investigation into the relationship between mutations and depression in a clinical sample of psychiatric patients from Albany Medical College. We were especially interested in the interplay between mutations of the GR and MR influencing cortisol levels and response in our bodies. DNA from patients was collected with buccal swabs and genotypes were analyzed through allele specific and quantitative polymerase chain reactions. We compared genotypes to the Beck Depression Inventory (BDI) score of patients, as well as other scales measuring anxiety and mood. No significant two-tailed correlation between individual SNPs or GR/MR SNP combinations and Genetic Predisposition to Depression iii BDI scores was found. One SNP in the GR gene (rs33389) and one SNP on the 11ßhydroxysteroid dehydrogenase type 1 (rs12086634), an enzyme that regulates peripheral cortisol levels, demonstrated significant one-tailed correlations with the Mindful Attention Awareness Scale and State Trait Anxiety scale, respectively. Obtaining a greater sample size would yield more representation of mutant alleles and potentially allow for more significance correlating genetic mutations to the incidence of depression. Genetic Predisposition to Depression 1 The Role of Single Nucleotide Polymorphisms Causing a Dysregulation of the HPA Axis on the Incidence of Depression In 2017, an estimated 262 million people, or 3.4% of the global population, were diagnosed with depression, making it one of the most prevalent diseases worldwide. In the United States alone, depression is the prominent causal factor of disability for people aged 15-44 (Ritchie and Roser, 2018). Depression manifests itself through a variety of different symptoms (figure 1), and the number, severity, and duration of symptoms can vary widely between patients. Multiple factors can contribute to the onset of depression, including personality traits and environmental factors, though most changes brought on by depression are a result of altered biochemical processes in the body (Torres, 2020). A prominent theory is that depressive symptoms are a result of our body’s repeated response to stress; when stressors become continuous or consistent over long periods of time is when depressive symptoms tend to arise (Deak, 2016). Figure 1. Symptoms of Major Depression. Taken from Cohen, 2020. Genetic Predisposition to Depression 2 Stress is regulated in our bodies by the Hypothalamic Pituitary Adrenal (HPA) axis, and its effector hormone, cortisol, is commonly labeled as the stress hormone (Deak, 2016). The HPA axis is composed of three cell populations in the hypothalamus, pituitary, and adrenal glands that communicate through the release of hormones, ultimately having metabolic effects on the body (Figure 2). When an environmental stressor is present, corticotropin releasing factor (CRF) neurons in the hypothalamus receive neural input from multiple brain regions, causing them to increase activity and produce and secrete the CRF hormone. This CRF hormone then travels to the pituitary gland and induces activity in corticotroph endocrine cells in the anterior pituitary. These cells produce and release adrenocorticotropic hormone (ACTH). ACTH migrates to the adrenal cortex where it binds to the melanocortin 2 receptor, inducing the conversion of cholesterol to cortisol through a sequence of enzyme-mediated reactions. The HPA axis is regulated through a series of negative feedback loops (Deak, 2016). Genetic Predisposition to Depression 3 Figure 2. Simplified diagram of the HPA axis and its components. CRF neurons in the hypothalamic paraventricular nucleus receive neural input due to some kind of stress, triggering the production and release of the CRF hormone, which travels to the pituitary gland. Corticotrophs in the pituitary gland secrete ACTH, and endocrine cells in the adrenal gland secrete cortisol. Cortisol is released into systemic circulation, reaching cells throughout the body and brain. Cortisol also induces direct negative feedback inhibition to the pituitary gland and hypothalamus. Taken from Endocrine Physiology, Fourth Edition (Molina, 2013). Cortisol is a glucocorticoid and the main effector hormone of the HPA axis. Glucocorticoids are steroid hormones that are involved in multiple physiological functioning’s necessary for daily life. Glucocorticoids’ main function is the regulation of metabolism, and secondary functions include the control of the inflammatory response and regulation of cognitive functions such as learning and memory (Gomez-Sanchez and Gomez-Sanchez, 2014; Timmermans et al., 2019). Cortisol release impacts metabolism by increasing blood pressure as well as increasing blood sugar by stimulating gluconeogenesis, and suppresses the immune response (Timmermans et al., 2019). Cortisol is the human glucocorticoid and exerts its effects through glucocorticoid and mineralocorticoid receptors (GR and MR receptors, respectively). The GR and MR are intracellular receptors that function as transcription factors after ligand binding. In their unbound form, the GR and MR are part of a multiprotein complex located in the cytoplasm, including chaperone proteins and immunophilins. Once cortisol binds, the receptors undergo conformational changes leading to dissociation from the protein complex and migration into the nucleus, where they regulate gene expression (Deak 2016; Moraitis et al., 2016). The GR is abundantly expressed in most cell types across the body, while the MR is expressed Genetic Predisposition to Depression 4 predominantly in only the hippocampus, amygdala, prefrontal cortex, and kidneys (Koning et al., 2019; Deak, 2016). The MR has a high affinity for glucocorticoids and is normally bound at low basal levels of cortisol, whereas the GR has a low affinity for glucocorticoids and is normally occupied only under elevated cortisol levels (such as due to a stressor), and is therefore more responsive to small changes in glucocorticoid concentrations (DeRijk et al, 2006; Chen et al., 2016; Deak, 2016). The activation of GR and MR are crucial for normal responses to stress; the receptors control processes regulating cognition, mood, and behavioral responses (Koning et al., 2019). When stressed, the activated MR aides in retrieving memory and appraisal processes, while the activated GR promotes consolidation of memory as well as energy redistribution to stop the stress response (Koning et al., 2019; Chen et al., 2016). Subsequently, proper functioning of the HPA axis is necessary for appropriate responses to stress. Dysregulations in the HPA axis, specifically in the activity of GR, MR, and circulating cortisol, have been theorized to contribute to improper responses to stress, and consequently, depression (Pariante, 2010; DeRijk et al, 2006; Chen et al., 2016; Plieger et al., 2017; Moraitis et al., 2016). This hypothesis is partially based on diseases of underand over-production of cortisol, Addison’s Disease and Cushing’s Syndrome, respectively (Figure 3). Genetic Predisposition to Depression 5 Figure 3. Common symptoms of Addison’s and Cushing’s syndrome. Addison’s Disease is characterized by insufficient production of aldosterone and cortisol, and symptoms can include irritability and depression (Rare Diseases, 2018). On the other end of the spectrum, Cushing’s syndrome is characterized by excess cortisol production by the adrenal glands, and presents symptoms including mood dysregulation and depression (Pariante, 2010; Tang et al., 2013; Chen et al., 2016). These disease states demonstrate that differences in circulating cortisol, hypo or hypercortisolemia, can lead to depressive symptoms. Many patients with psychiatric disorders have been shown to express diminished HPA axis negative feedback regulation, demonstrated by elevated cortisol levels (Pariante, 2010). Unlike Cushing’s and Addison’s, in which elevated or reduced cortisol levels affect brain function through normally working GR/MR receptors, studies into depression exhibit altered cortisol responsiveness due to impaired functioning receptors (Pariante, 2010). Pariante and Miller measured GR function in vitro and in vivo and found that depressed patients have a diminished GR response when compared to healthy controls (2001). This diminished GR capacity is hypothesized to arise from either impaired function or decreased expression of GR in the brain (Pariante, 2010). Impaired Genetic Predisposition to Depression 6 GR functioning is commonly referred to as glucocorticoid resistance, as the GR is unable to respond effectively to circulating cortisol, and this leads to elevated cortisol levels

Follicular helper T (Tfh) cells promote, whereas follicular regulatory T (Tfr) cells restrain, germinal center (GC) reactions. However, the precise roles of these cells in the complex GC reaction remain poorly understood. Here, we perturb Tfh or Tfr cells after SARS-CoV-2 spike protein vaccination in mice. We find that Tfh cells promote the frequency and somatic hypermutation (SHM) of Spike-specific GC B cells and regulate clonal diversity. Tfr cells similarly control SHM and clonal diversity in the GC but do so by limiting clonal competition. In addition, deletion of Tfh or Tfr cells during primary vaccination results in changes in SHM after vaccine boosting. Aged mice, which have altered Tfh and Tfr cells, have lower GC responses, presenting a bimodal distribution of SHM. Together, these data demonstrate that GC responses to SARS-CoV-2 spike protein vaccines require a fine balance of positive and negative follicular T cell help to optimize humoral immunity.