IL-1β is typically associated with the innate response, often produced following the detection of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). It is also identified as a cytokine involved in bridging the innate and adaptive immune responses, in which innate cells, like dendritic cells, use IL-1β to activate T cells for the adaptive immune response. The role of IL-1 signaling has been established in the context of T cell differentiation and function, particularly in its regulation of T follicular helper (TFH) cells. Recent work has demonstrated that with TFH function primarily acting within the germinal center (GC), a local source of IL-1β must be present, identifying GC B cells as a critical source of IL-1β. Here we discuss the roles of cells within the GC milieu, the cytokines they produce, and their impact on the GC. In addition, we also discuss the findings from studies examining the molecular mechanisms underlying IL-1β production in B cells and its impact on B cell function. This review is especially relevant as it draws together findings from various disease pathologies to consolidate our current understanding of B cell subsets producing IL-1β and IL-1β signaling within the GC, in both T cells and B cells.

Influenza infected mice with a B cell-specific interleukin-1β (IL-1β) deficiency exhibit significant reduction in germinal center (GC) B cells, T follicular helper cells (TFH), and impaired trafficking of TFH cells into the B cell follicles, delineating a role of B cell-derived IL-1β in the GC reaction. Two signals drive IL-1β production - 1) NFkB mediated pro-IL1β expression and 2) activation of inflammasomes to cleave pro-IL1β into the active IL-1β. Extracellular ATP, ROSs, and ion fluxes within the GC suggest the activation of the NOD-like receptor-3 (NLRP3) inflammasome. We hypothesized that NLRP3 activity mediates IL-1β production in GC B cells. Our data show that GC B cells had significantly higher expression of NLRP3 than non-GC (nGC) and naïve B cells. B cell-specific ablation of NLRP3, but not of other inflammasome sensor proteins, showed significant reductions in GC B cell and TFH cell numbers. Mechanisms underpinning NLRP3 activity, like active caspase-1 (aCasp-1) and caspase-8 (aCasp-8) are upregulated in GC B cells. However, coexpression analysis showed significantly higher coexpression of aCasp-1 with IL-1β compared to aCasp-8 with IL-1β in GC vs. nGC B cells, suggesting aCasp-1 is mediating IL-1β activation. Similar to murine B cells, human activated GC B cells from the palatine tonsils showed IL-1β production, mediated by aCasp-1 and NLRP3 inflammasome activity. Our findings expose novel molecular targets to enhance the GC response, post influenza infection. Immune Response Regulation: Cellular Mechanisms (IRC)

Persistent germinal center (GC) responses show increased benefit in optimal responses to influenza infection. Follicular helper T (TFH) cells provide the essential signals and help for maintenance of GCs and require IL-1β signaling for establishment and maintenance. We observe a preferential upregulation of IL-1β within GC B cells and coexpression of NLRP3 and caspase-1 with IL-1β confirms that GC B cells process IL-1β using a canonical NLRP3/caspase-1 mechanism. Using B cell specific ablation of IL-1β production and IL-1β signaling we further confirm that, GC B cells are the primary source of vital IL-1β within the GC and that IL-1β processing by GC B cells post influenza infection is driven by NLRP3 inflammasomes. We observe significant reduction of GC B cells and TFH cells in the absence of B cell derived IL-1β and our analysis of human B cells suggests similar mechanisms in human GC B cells. Our data present GC B cells in two novel roles, the first in producing IL-1β, which is associated with innate functions, within the GC and the second is providing helper cytokine to the TFH cell. Our findings add to the known complexity of the GC providing a target to enhance GC function and persistence.

While human and mouse memory B cells (MBCs) can express the transcription factor T-bet, its role in regulating MBC function remains unclear. We characterized multiple transcriptionally distinct clusters of mature, somatically mutated nucleoprotein (NP)-specific MBCs in lymph nodes (LNs) and lungs of influenza-infected mice. Although none of the MBCs expressed the plasma cell (PC) lineage commitment factor Blimp1, one cluster was enriched for Tbx21+ cells. Similar to the previously described human T-bet+ effector MBC (eMBC) population, Tbx21+ mouse MBCs upregulated gene networks associated with effector metabolism, protein synthesis, and the unfolded protein response. Constitutive and inducible ablation of T-bet in murine B cells showed that T-bet expression by MBCs was required for persistence of LN and lung eMBCs with rapid in vitro and in vivo PC differentiation potential. Thus, T-bet marks NP+ eMBCs that are poised to differentiate, and it regulates maintenance of lung-resident MBCs and local PC responses following virus re-exposure.

Although the transcription factor (TF) T-bet is expressed by antigen-experienced human B cells, including the extrafollicular IgD negCD27 negmemory-like DN2 cells and the IgD negCD27 +effector memory cells (eBmem), it is unknown whether T-bet expression influences the development, maintenance, or function of Bmem cells. Using oligo-labeled recombinant influenza nucleoprotein (NP) tetramers and paired single cell RNA and V(D)J sequencing, we characterized NP-specific Bmem in the draining LN of flu-infected memory mice. We identified 7 transcriptionally distinct clusters of mature non-replicating β Bmem cells. While none of the Bmem clusters expressed TFs required for plasma cell (PC) lineage commitment, one cluster, containing clonally-expanded somatically-mutated Bmem cells, expressed significantly higher levels of the T-bet gene, Tbx21, and was transcriptionally very similar to human T-bet +eBmem. The NP +Tbx21 +Bmem upregulated gene networks associated with metabolic reprogramming, protein synthesis and the mTOR-dependent unfolded protein response, suggesting that these cells might represent Bmem that are metabolically poised for rapid PC differentiation. Using constitutive and inducible models to delete Tbx21 specifically in B cells we showed that the rapid differentiation potential of Bmem is controlled by T-bet and that T-bet is required for the development and persistence of NP +eBmem subsets in LN and lung. Thus T-bet not only marks Bmem with effector potential but also regulates the persistence and function of flu-induced NP +Bmem that are transcriptionally poised to provide rapid humoral protection from infection. Supported by a grant from the NIH (R01 AI110508)

B cells and the antibodies they produce are essential components of immunity to respiratory viruses, including influenza virus (IAV) and SARS-CoV-2. B cells responding to viral infection can differentiate into antibody secreting cells (ASCs), or resting memory B cells, some of which are tissue-resident (BRM cells). ASCs provide protection by secreting virus-specific antibodies, whereas BRM cells rapidly differentiate into ASCs after secondary infection. We previously published that IAV-specific BRM cells are retained in the lungs and do not recirculate. We now show that many lung-resident BRM cells reside in the airways and can be collected by bronchoalveolar lavage (BAL). BRM cells in the BAL are phenotypically and functionally different than those in the lung tissue and lymph nodes and express markers at various levels that possibly effect function. There is also a distinction in the role of the ASCs between the lungs and the BAL that will secrete certain antibodies at different time points during the IAV challenge. To understand BRM cell homing, differentiation, and function, we are using single cell approaches to define the transcriptional and epigenetic programs of airway-resident BRM cells at resting memory and during the response to IAV challenge. We are also using this technique to identify the changes among the entire immune system during a secondary IAV challenge to gain an understanding of how the entire immune system plays a role in protection. By understanding the programs that regulate BRM placement in the lung and their response to challenge infection, we expect to identify ways to improve vaccines against respiratory viruses and ameliorate B cell-dependent lung pathology in the context of autoimmunity or allergy. Supported by grants from NIH (R01 AI152476)

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

In mammals, adaptive immunity is mediated by a broadly diverse repertoire of naive B and T lymphocytes that recirculate between secondary lymphoid organs. Initial antigen exposure promotes lymphocyte clonal expansion and differentiation, including the formation of memory cells. Antigen-specific memory cells are maintained at higher frequencies than their naive counterparts and have different functional and homing abilities. Importantly, a subset of memory cells, known as tissue-resident memory cells, is maintained without recirculating in nonlymphoid tissues, often at barrier surfaces, where they can be reactivated by antigen and rapidly perform effector functions that help protect the tissue in which they reside. Although antigen-experienced B cells are abundant at many barrier surfaces, their characterization as tissue-resident memory B (BRM) cells is not well developed. In this study, we describe the characteristics of memory B cells in various locations and discuss their possible contributions to immunity and homeostasis as bona fide BRM cells.

Memory B cells are found in lymphoid and non-lymphoid tissues, suggesting that some may be tissue-resident cells. Here we show that pulmonary influenza infection elicited lung-resident memory B cells (BRM cells) that were phenotypically and functionally distinct from their systemic counterparts. BRM cells were established in the lung early after infection, in part because their placement required local antigen encounter. Lung BRM cells, but not systemic memory B cells, contributed to early plasmablast responses following challenge infection. Following secondary infection, antigen-specific BRM cells differentiated in situ, whereas antigen-non-specific BRM cells were maintained as memory cells. These data demonstrate that BRM cells are an important component of immunity to respiratory viruses such as influenza virus and suggest that vaccines designed to elicit BRM cells must deliver antigen to the lungs.

Through a combination of fluorescence microscopy and patch-clamp analysis we have identified a striking alteration in K+ channel expression in terminally differentiated human CCR7–CD45RA– effector memory T lymphocytes (TEM). Following activation, TEM cells expressed significantly higher levels of the voltage-gated K+ channel Kv1.3 and lower levels of the calcium-activated K+ channel IKCa1 than naive and central memory T cells (TCM). Upon repeated in vitro antigenic stimulation, naive cells differentiated into Kv1.3highIKCa1low TEM cells, and the potent Kv1.3-blocking sea anemone Stichodactyla helianthus peptide (ShK) suppressed proliferation of TEM cells without affecting naive or TCM lymphocytes. Thus, the Kv1.3 highIKCa1low phenotype is a functional marker of activated TEM lymphocytes. Activated myelin-reactive T cells from patients with MS exhibited the Kv1.3highIKCa1low TEM phenotype, suggesting that they have undergone repeated stimulation during the course of disease; these cells may contribute to disease pathogenesis due to their ability to home to inflamed tissues and exhibit immediate effector function. The Kv1.3highIKCa1low phenotype was not seen in glutamic acid decarboxylase, insulin-peptide or ovalbumin-specific and mitogen-activated T cells from MS patients, or in myelin-specific T cells from healthy controls. Selective targeting of Kv1.3 in TEM cells may therefore hold [...] Article Autoimmunity

Abstract B cells display phenotypic and functional heterogeneity in multiple anatomical locations following vaccination or infection. Influenza-specific memory B cells (Flu+BMEM cells) are found in both lymphoid tissues and lung. It is unclear whether these cells represent circulating or resident memory B cell (BRM) populations. We hypothesized that a portion of the Flu+BMEM cell population in the lung would be non-circulating, BRMs. To determine whether Flu+BMEM cells in the lung are circulating or resident, we parabiotically joined previously-infected, congenically-mismatched mice for 2 weeks and marked those cells currently in circulation by infusing anti-B220 and identified those that have trafficked between the partners by their expression of the CD45 congenic marker. The lungs of these mice had large populations of hemagglutinin and nucleoprotein -specific BMEM cells that did not attain equilibrium within 2 weeks of parabiosis, suggesting that they are non-circulating. The Flu+ BRMs in the lungs consisted of 56% IgM+ and 43.9% isotype-switched BRMs. They were established as early as 15 days after infection and maintained for at least 60 days. The formation of Flu+ BRMs required the germinal center (GC), as blocking CD40L with MR1 antibody, during the primary infection abrogated BRM. However, MR1-treatment of mice with established BRM did not affect BRMs in the lung, even though Flu+ GC B cells could be detected in the LN for up to 90 days. These data suggest that GC-dependent lung-BRMs are established early after infection and maintained independently of GCs. These findings are important in the development of vaccines that elicit BRMs and they will provide mechanistic information into the function of Ag+BMEM cells residing in the mucosa.

A plethora of work implicates important effects of the vitamin A derivative retinoic acid (RA) in myeloid differentiation, whereas fewer studies explore the role of RA in lymphoid cells. Most work on lymphoid cells has focused on the influence of RA on CD4 T cells. Little information about the role of RA in CD8 T cell differentiation is available, and even less on cell-intrinsic effects in the CD8 T cell. This study explores the role of RA in effector and memory differentiation in a cell-intrinsic manner in the context of vaccinia virus infection. We observed the loss of the short-lived effector cell phenotype (reduced KLRG1(+), T-bet(hi), granzyme B(hi)), accompanied by an enhanced memory precursor phenotype at the effector (increased CD127(hi), IL-2(+)) and contraction phases (increased CD127(hi), IL-2(+), eomesodermin(hi)) of the CD8 response in the absence of RA signaling. The lack of RA also increased the proportion of central memory CD8s. Collectively, these results introduce a new role for RA in CD8 T cell activation and differentiation. This new role may have significant implications for optimal vaccine design in which vitamin A supplementation is used to augment effector responses, but it may be to the detriment of the long-term central memory response.

ABSTRACT MicroRNAs are key regulators of the immune response, but their role in CD8 T cell differentiation in vivo is not known. We show that miR-155 is important in both effector and memory antiviral CD8 T cell responses. Without miR-155, there was a weaker effector response and a skewing toward memory precursor cells. At the memory stage, miR-155-deficient CD8 T cells preferentially differentiated into central memory cells and were capable of mounting a potent secondary response.

A plethora of work implicates important effects of the vitamin A derivative retinoic acid (RA) in myeloid differentiation, whereas fewer studies explore the role of RA in lymphoid cells. Most work on lymphoid cells has focused on the influence of RA on CD4 T cells. Little information about the role of RA in CD8 T cell differentiation is available, and even less on cell-intrinsic effects in the CD8 T cell. This study explores the role of RA in effector and memory differentiation in a cell-intrinsic manner in the context of vaccinia virus infection. We observed the loss of the short-lived effector cell phenotype (reduced KLRG1 + , T-bet hi , granzyme B hi), accompanied by an enhanced memory precursor phenotype at the effector (increased CD127 hi , IL-2 +) and contraction phases (increased CD127 hi , IL-2 + , eomesodermin hi) of the CD8 response in the absence of RA signaling. The lack of RA also increased the proportion of central memory CD8s. Collectively, these results introduce a new role for RA in CD8 T cell activation and differentiation. This new role may have significant implications for optimal vaccine design in which vitamin A supplementation is used to augment effector responses, but it may be to the detriment of the long-term central memory response. T he morphogenic role of all-trans retinoic acid (RA), a vitamin A derivative, in development and differentiation was confirmed by White et al. (1) in 2007, using a zebra fish model to confirm RA patterning in the hindbrain. However, immunologists have studied it in various contexts of immune cell differentiation as early as the 1980s. Among myeloid cells, RA has been shown to allow for differentiation into mature macro-phages or APCs. (2). This RA-mediated differentiation of den-dritic cells (DCs) has been shown to skew them toward IL-12– producing DCs (3). RA also regulates isotype switching and plasma cell formation by B cells (4–6). In the adaptive immune compartment, RA has been shown to promote regulatory CD4 T and CD8 T cell differentiation and stabilization (7–9). Further, RA has been shown to enhance in-flammatory effector responses by CD4 helper T cells (10, 11). In CD8 T cells, an early study showed that increased expression of RA receptor g increased the number of CD8 T cells (12). To our knowledge, no previous studies have looked at the cell-intrinsic role of RA signaling in CD8 T cell effector and …

We investigated CD45RA and CCR7 expression in CD4+ and CD8+ subsets of cerebrospinal fluid (CSF) lymphocytes, both immediately ex vivo and after stimulation, from 134 patients with a variety of inflammatory and non-inflammatory neurological diseases. Most inflammatory diseases had a higher CD4+:CD8+ ratio and higher percentage of effector memory T cells (T(EM)) than non-inflammatory controls, excluding active infection. Moreover, we found that patients with highly elevated cell counts in the CSF tended to have a lower percentage of central memory T cells (T(CM)) than patients with low or absent pleocytosis, with a concomitant increase in T(EM). We also found that samples with elevated IgG index or presence of oligoclonal bands had a significantly higher CD4+:CD8+ ratio than normal samples, consistent with increased CD4+ help for intrathecal IgG synthesis by B cells.

A link between autoimmunity and improved antitumor immunity has long been recognized, although the exact mechanistic relationship between these two phenomena remains unclear. In the present study we have found that vitiligo, the autoimmune destruction of melanocytes, generates self antigen required for mounting persistent and protective memory CD8+ T cell responses to melanoma. Vitiligo developed in approximately 60% of mice that were depleted of regulatory CD4+ T cells and then subjected to surgical excision of large established B16 melanomas. Mice with vitiligo generated 10-fold larger populations of CD8+ memory T cells specific for shared melanoma/melanocyte antigens. CD8+ T cells in mice with vitiligo acquired phenotypic and functional characteristics of effector memory, suggesting that they were supported by ongoing antigen stimulation. Such responses were not generated in melanocyte-deficient mice, indicating a requirement for melanocyte destruction in maintaining CD8+ T cell immunity to melanoma. Vitiligo-associated memory CD8+ T cells provided durable tumor protection, were capable of mounting a rapid recall response to melanoma, and did not demonstrate phenotypic or functional signs of exhaustion even after many months of exposure to antigen. This work establishes melanocyte destruction as a key determinant of lasting melanoma-reactive immune responses, thus illustrating that immune-mediated destruction of normal tissues can perpetuate adaptive immune responses to cancer.

The T cell response possesses a number of inhibitory receptors to regulate the extent of the antiviral response and prevent immune pathology. These receptors are generally transiently upregulated during an effector response and then downregulated during memory. Some inhibitory receptors, such as programmed death 1 (PD-1) and LAG-3, were shown to be aberrantly upregulated during memory to chronic lymphocytic choriomeningitis virus infection, limiting functional capabilities. However, little is known about the impact of inhibitory receptors on memory development during a normal CD8 T cell response to acute virus infection. Our previous data showed that PD-1 is aberrantly upregulated during a secondary response by memory CD8 T cells that were generated without CD4 T cell help. Therefore, we examined the role of PD-1 in memory differentiation during acute vaccinia virus infection in intact mice. In the absence of PD-1, the primary and memory CD8 T cell responses were enhanced. Moreover, there were distinct phenotypic and functional changes in the memory PD-1(-/-) CD8 T cells. Higher levels of CD62L, CD27, and CCR7 were detected; cells produced more IL-2 and made an enhanced secondary response. These changes indicate a skewing of the memory population toward the central memory phenotype in the absence of PD-1 signaling.