CD169+ subcapsular sinus macrophages (SSMs) line the capsule of the lymph node and have important roles in antigen capture and display. To better understand the biology of these unique macrophages, multiple labs, including ours, have attempted to isolate these cells by flow cytometry. We demonstrated that many cells that stain positive for the macrophage marker CD169 are not macrophages, but rather correspond to a population of IL-17-committed lymphocytes that acquire macrophage-derived CD169+ membrane fragments. These IL-17-committed IL-7Ra+CCR6+ lymphocytes were present at the lymph node subcapsular sinus and migrated in close association with CD169+ SSMs. We suggest that crosstalk between SSMs and IL-17-committed lymphocytes may promote lymph node barrier immunity.

Innate-like IL-17-committed lymphocytes are an important source of IL-17 in a variety of infections and autoimmune diseases. Building on our observation that these cells, including IL-17-committed gdT cells (gdT17), are enriched in skin-draining lymph nodes, we asked whether a related gdT cell population was present in the skin and subsequently identified a population of IL-7Ra+ CCR6+ RORgt+ gdT cells resident in the mouse dermis. Intravital imaging revealed that gdT cells migrate in the dermis, while epidermal gdT cells are stationary. Dermal gdT cells rapidly produced IL-17A in vitro following exposure to IL-1b and IL-23. We suggest that dermal gdT17 cells are an important source of IL-17A during the early stages of cutaneous infection.

Finally, we made the fortuitous observation that a commonly used congenic CD45.1+ C57BL/6 mouse substrain is characterized by a selective deficiency for the Vg4+ subset of gdT17 cells. We identified a spontaneous mutation in the transcription factor Sox13 in this strain, and suggest that Sox13 is intrinsically required for the development of Vg4+ gdT17 cells. In a mouse model of psoriasis-like dermatitis, we observed a dramatic expansion of Vg4+ gdT17 cells in the draining lymph node followed by their accumulation in inflamed skin. Vg4+ gdT17 cells selectively homed to inflamed skin following transfer, suggesting that these cells may recirculate from lymph nodes to inflamed peripheral tissues. Sox13 mutant mice, which lack Vg4+ gdT17 cells, are protected from psoriasis-like changes in this model of skin disease.

Regulatory T cells (Tregs) are critical to the maintenance of immune homeostasis and the prevention of autoimmunity. In the NOD mouse model of autoimmune diabetes, treatment with therapeutic Tregs can prevent and even reverse disease onset. The means by which Tregs are able to exert their protective effects in vivo have remained incompletely understood. This work focused on elucidating the critical cellular targets of Tregs at the site of inflammation and the effects of Tregs on these target cells. This was accomplished by examining the target tissue, the pancreatic islets of Langerhans, before and after Treg treatment with a variety of approaches. Islets and their immune infiltrates were analyzed at the transcriptional and post-transcriptional levels. Islet immune infiltrates were characterized at the phenotypic and functional levels by flow cytometry and by multiple modes of microscopic imaging. Therapeutic Tregs were found to quickly home to inflamed islets, where they engaged in dynamic interactions with resident dendritic cells (DCs). Treg arrival was followed by a rapid decrease in islet CD8 T cells that corresponded with a reduction in the expression of cytotoxic effectors and cytokines. T cells that persisted in the islets following Treg treatment showed no decrease in proliferative capacity but were suppressed at the level of effector function, where they lacked production of IFNg protein despite continued expression of IFNg mRNA. In CD8 T cells, this inhibition of effector function was related to a decrease in mTOR signaling that was required for sustained IFNg production. In addition to these acute effects on CD8 T cells following Treg treatment, we also observed a more gradual impact on islet DCs. The recruitment of DCs was reduced in the weeks following Treg treatment. Islet DCs were found to arise from a blood monocyte precursor. Overall, our findings that therapeutic Tregs target CD8 T cells during acute disease control and DCs during later stages implicate the importance of these targets in disease pathogenesis and elucidates the mechanisms by which therapeutic Tregs exert their protective effects.

Basophils represent a rare cell population involved in allergic responses and are one of the primary effector cells for IgE-induced inflammation. However, the basis for which certain IgE-antigen interactions trigger basophil activation and the downstream mechanisms for basophil-enhanced inflammation remain unclear. We examined how IgE affinity and overall antigen-IgE/Fc epsilon receptor I (FcεRI) binding differentially relate to basophil activation. Using IgE-loaded murine basophils, we observed that high affinity antigens promoted enhanced basophil degranulation and IL-4 production compared to low affinity antigens, even when the relative concentrations of these antigens were adjusted to achieve the same equilibrium binding to IgE/FcεRI. Similarly, antigen exposure of basophils in the lymph node also lead to enhanced basophil degranulation using high affinity antigen compared to low affinity antigen after adjusting for antigen surface binding. Subcutaneous exposure of high affinity antigen, but not low affinity antigen, promoted systemic basophil activation at distal sites and increased susceptibility to anaphylaxis. We propose that the affinity of IgE for antigen is a primary determinant of basophil degranulation and is a crucial factor in susceptibility to severe IgE-mediated responses such as anaphylaxis.

We also determined whether basophils promote IgE-induced responses in the lymph node. Exposure to a variety of stimuli leads to the accumulation of basophils into the draining lymph node. There, basophils have been proposed to act as critical initiators of the type II adaptive immune response, though more recent reports have challenged this hypothesis. Since then, a clear role for basophils in entering the lymph node has remained elusive. We investigated an alternative function for basophil accumulation into the lymph node: to participate in a local allergic inflammatory response. Through histological examination, basophils were observed to closely associate with dense inflammatory cell clusters comprised of eosinophils, alternatively activated macrophages, multinucleated giant cells, and a reorganized stromal cell population. These cell clusters increased upon antigen rechallenge. Testing basophil-deficient mice revealed that basophils were important contributors in both inflammatory cell recruitment and cell cluster formation. Moreover, IgE-induced activation of basophils was sufficient to promote the allergic inflammatory response in the lymph node. Behaviorally, basophils were observed to respond to incoming antigen by degranulating, producing IL-4, relocalizing closer to sites of antigen entry, and increasing in extended interactions with macrophages, suggesting important steps in the development of localized basophil-mediated responses. Finally, basophils associated with allergic inflammatory cell clusters that had formed in response to lymph node infiltration by the lymphatic-traversing helminth, Brugia pahangi, revealing the targeted and potentially defensive nature of the immune response. Altogether, we propose that a primary function for basophil entry into the lymph node is to participate in and promote a local allergic inflammatory response to protect against a perceived threat.

T cells undergo dynamic and rapid changes in gene expression and protein output in response to cognate antigen, processes aided by post-transcriptional regulation. RNA binding proteins (RPBs) are important trans factors that bind to the 3’ untranslated region (3’UTR) of transcripts and modulate mRNA degradation and translation. Loss of function of these proteins, as well as mutations in their cis-regulatory binding sites, can alter T function and development and lead to immune-mediated diseases. While studies have characterized the network of RBPs present in T cells, as well as binding profiles of individual RBPs, the cis-regulatory landscape of the T cell transcriptome is yet to be defined. This body of work addresses this gap by developing a biochemical assay to map the RBP binding profile in T cells and identify post-transcriptional cis-regulatory regions that regulate protein expression and T cell function. Using our technique, GCLiPP, we identified biochemically shared binding sites across conserved regions of human and mouse 3’UTRs that govern transcript stability. Additional dissections of predicted RBP binding sites in several 3’UTRs of immune-related genes revealed cis-regulatory regions critical for modulating protein expression. We performed further analysis on CD69, a negative regulator of T cell egress, and identified a conserved destabilizing region within the 3’UTR. Absence of this region, as well as the whole 3’UTR, increased CD69 transcript half-life and led to higher CD69 expression as well as more CD69+ T cells in mouse and human. The post-transcriptional circuitry within Cd69 3’UTR had a profound effect on T cell migration, as mice with homozygous deletion of this region showed impaired thymic egress. Taken together, our studies provide a resource for mapping post-transcriptional cis-elements in the T cell transcriptome and demonstrate its utility for discovering functional cis-regulatory regions that contain variants associated with human immune-mediated diseases.

During infection, pathogen-specific T cells must efficiently recognize their cognate antigen, expand into effector cells, and form memory cells that provide protection during subsequent encounters with the same pathogen. Chemokines and chemokine receptors play essential roles in these processes. They mediate T cell surveillance of antigens in secondary lymphoid organs (SLOs) and facilitate T cell contacts with other immune cells that result in activation, expansion into effector cells, and formation of functional memory cells. Within the spleen, activated CD8 T cells move from distinct compartments of the white pulp (WP) to the marginal zone (MZ) and red pulp (RP), where they exit the spleen and migrate to inflamed tissues. The molecular cues that guide activated CD8 T cells to splenic compartments and the effect of these movements on T cell differentiation are not completely understood. The chemokine receptor CXCR3 is expressed on activated CD8 T cells and guides these cells towards the interferon-inducible chemokines CXCL9, CXCL10, and CXCL11. We used wild-type (WT) and CXCR3 knock-out (KO) T cell receptor (TCR) transgenic P14 CD8 T cells to determine if CXCR3 directs activated CD8 T cells to specific splenic compartments and if CXCR3-directed movements influence CD8 T cell differentiation. Using lymphocytic choriomeningitis virus (LCMV) infection, we found that CXCR3 directed activated CD8 T cells towards CXCR3 ligands in the MZ, where LCMV antigens were abundant. In contrast, CXCR3 KO CD8 T cells were concentrated in the T zone away from antigen in the MZ. Because antigen and inflammation promote the generation of short-lived effector cells, we examined whether CXCR3-mediated positioning affected CD8 T cell differentiation. CD8 T cell clustering in the MZ was associated with increased generation of short-lived effector cells and decreased formation of memory precursor cells. This led to the generation of fewer memory cells that were phenotypically and qualitatively different. These data demonstrate that CXCR3 increases CD8 T cell access to antigen and potentially inflammation in the MZ that affects the balance between effector and memory cell generation.

The immune system is organized to allow lymphocytes to survey for antigen and rapidly respond to an infection. B lymphocytes reside in B cell follicles and, upon exposure to antigen, rapidly migrate to interact with T cells, and then later go on to quickly produce protective antibodies and more slowly generate a memory response to prevent later reinfection. The role of chemokines and their receptors in positioning naïve and activated cells is well appreciated; CXCR5 is required for B cells to home to B cell follicles, and CCR7 is required for migration toward T cells. However, the positioning of B cells is not completely explained by these two chemokine receptors. During activation, B cells migrate after T cell interaction to the outer follicle, and some then migrate toward the center of the follicle to form germinal centers. The cues positioning cells to these areas have been poorly understood. Here we describe the role of the G protein coupled receptor EBI2 in positioning B cells, both homeostatically and upon activation. EBI2 promotes naïve B cell localization to the outer follicle, and EBI2 deficient cells are found toward the center of the follicle in competition. EBI2 expression is increased after B cell activation, directing activated cells to the outer follicle before CCR7 brings them toward T cells. After receiving CD40 stimulation from T cells, activated B cells migrate again to the outer follicle in an EBI2-dependent manner. EBI2 downregulation is required for B cells to move into the follicle and for germinal center development. Some cells remain at the outer follicle and produce antibody. EBI2 is required for efficient antibody production in a T-dependent immune response. These data demonstrate that EBI2 acts in coordination with the chemokine receptors CXCR5 and CCR7 to position naïve and activated B cells and contributes to the immune response.